De novo surface preparation and uses thereof

ABSTRACT

Methods and formulations for preparing low non-specific binding surfaces are described, and the prepared surface can provide improved performance for nucleic acid detection and base calling applications. The surface provides more accurate nucleic acid detection, enhanced contrast to noise ratio, and better data collection.

CROSS REFERENCE

This application is a continuation of PCT/US2019/061556, filed Nov. 14,2019, which is a continuation-in-part of U.S. application Ser. No.16/363,842, filed Mar. 25, 2019, which claims the benefit of U.S.Provisional Application No. 62/767,343, filed on Nov. 14, 2018, and ofU.S. Provisional Application No. 62/776,898, filed on Dec. 7, 2018, eachof which is incorporated herein by reference in its entirety.

BACKGROUND

A variety of DNA sequencing methodologies have been developed andcommercialized over the past two decades (see, for example, E. Mardis(2008), “Next-Generation DNA Sequencing Methods”, Annu. Rev. GenomicsHum. Genet. 9:387-402; and J. Heather and B. Chain, (2016), “TheSequence of Sequencers: The History of Sequencing DNA”, Genomics 107:1-8for recent reviews). Many “second generation” and “third generation”sequencing technologies utilize a massively parallel, cyclic arrayapproach to sequencing-by-synthesis (SBS), in which accurate decoding ofa single-stranded template oligonucleotide sequence tethered to a solidsupport relies on successfully classifying signals that arise from thestepwise addition of A, G, C, and T nucleotides by a polymerase to acomplementary oligonucleotide strand. These methods typically requirethe oligonucleotide template to be modified with a known adaptersequence of fixed length, affixed to a solid support in a random orpatterned array by hybridization to surface-tethered probes of knownsequence that is complementary to that of the adapter sequence, and thenprobed using, for example, a single molecule (non-amplified),synchronous sequencing-by-synthesis (smSBS) approach (e.g., the Helicostechnology), or a single molecule, asynchronous sequencing-by-synthesis(smASBS) approach (e.g., the Pacific Biosciences technology). In thesmSBS approach, terminator nucleotides encoded with fluorescent tags areused, such that a replication enzyme can only incorporate a single baseper cycle. The Helicos technology, for example, used a singlefluorescent tag and a sequential introduction of A, G, C, T wasperformed—once base per cycle. During each cycle, an imaging step wasperformed to classify the correct ‘base” for each single moleculetemplate on an array. Following the imaging steps, the reversibly-linkedtags are removed, such that the replicating enzyme (polymerase) canincorporate the next templating base. These cycles are repeated manytimes to eventually decode the template oligonucleotide strands on therandom array and determine their respective sequences.

While successful, the cyclic array approach has generally suffered fromtwo fundamental inadequacies: (i) the cycle times for addition of eachsuccessive nucleotide to the complementary strand are long, and (ii) thesignals arising from the stepwise addition of single nucleotides areweak (typically detected through the use of fluorescent labels andfluorescence imaging techniques) and exhibit low contrast-to-noiseratios (CNRs) as will be discussed in more detail below, and thereforerequire long imaging times using costly instrumentation comprising highprecision optics to achieve accurate base-calling.

Attempts to address the cycle time issue for cyclic array sequencingapproaches have been made, for example, through the advent of singlemolecule asynchronous sequencing-by-synthesis (smASBS) approaches, e.g.,the Pacific Biosciences technology in which four spectrally-distinctfluorescent tags are linked to the respective A, G, C, and Tnucleotides, the addition of which can then be classified in“real-time”. In this approach, all four labeled nucleotides areintroduced simultaneously and images are acquired during the entirestrand replication process. Each position in the sequence is classifiedas ‘A’, ‘G’, ‘C’, and ‘T’ based on the spectrum of the detected light.Here, the cycle times can theoretically be as fast as thepolymerase-catalyzed replication rate, but the trade-off is decreasedCNR, thereby introducing classification errors that ultimately lead todiminished accuracy, and putting greater reliance on high precisionoptics and costly instrumentation.

Attempts to address the signal limitations in some cyclic arraysequencing approaches (i.e., non-single molecule approaches) have beenmade by incorporating an amplification step in the process. Solid-phaseamplification of template DNA molecules tethered to a solid support in arandom or patterned array increases the number of copies of the targetto be sequenced, such that the signal arising from a “colony” ofreplicate template molecules upon step-wise addition of detectable basesto their respective complementary strands can be classified as ‘A’, ‘G’,‘C’, or ‘T’. The probability of successful classification (and thus theaccuracy of base-calling) is dependent on the respective CNR during eachdetection event, which is often limiting.

Thus, there is a need for improved solid supports and solid phaseamplification methods for nucleic acid sequencing that will increase themagnitude of base addition signals, decrease non-specific backgroundsignals, and thus improve CNR, thereby improving the accuracy ofbase-calling, potentially shortening cycle times, and reducing thedependence of the sequencing process on high precision optics and costlyinstrumentation.

SUMMARY

Some embodiments relate to a method of performing nucleic acid sequencedetermination, the method comprising: a) providing a surface; whereinthe surface comprises: i) a substrate; ii) at least one hydrophilicpolymer coating layer; iii) a plurality of oligonucleotide moleculesattached to at least one hydrophilic polymer coating layer; and iv) atleast one discrete region of the surface that comprises a plurality ofclonally-amplified sample nucleic acid molecules immobilized to theplurality of attached oligonucleotide molecules, wherein the pluralityof immobilized clonally-amplified sample nucleic acid molecules arepresent with a surface density of at least 5000 molecules/mm², b)performing a nucleic acid amplification reaction on sample nucleic acidmolecules prior to or after annealing them to the plurality ofoligonucleotide molecules; and c) performing at least a singlenucleotide binding or incorporation reactions, wherein the nucleotidesare labeled with a detectable tag. In some embodiments, the methodfurther comprises detecting or characterizing the nucleotide based onthe detectable tag.

Disclosed herein are surfaces comprising a substrate, at least one layerof a hydrophilic, low nonspecific binding (i.e., low background)coating, and a plurality of oligonucleotide molecules attached to atleast one layer of hydrophilic, low-binding, low background coating.

The disclosed low nonspecific binding solid supports may be used for avariety of bioassays including, but are not limited to, DNA sequencingand genotyping. These supports comprise temperature- andchemically-stable functionalized substrates that withstand exposure tomultiple solvent exchanges and temperature changes, and that confer lownonspecific binding properties throughout the duration of the assay. Thedisclosed supports may have some or all of the following properties:

1. Surface functionalization performed using any combination of polarprotic, polar aprotic and/or nonpolar solvents that leads to an increasein the efficacy of bioassay performance by >5-fold (e.g., improvement inreaction rate and/or desired product formation, respectively) overtraditional approaches.

2. Minimal contact angle measurement post functionalization (e.g., <35degrees), which is maintained through successive solvent and temperaturechanges.

3. Low nonspecific binding of biomolecules versus specifically-boundmolecules (e.g., greater than 1 specifically-bound molecule vs.<0.25nonspecifically-bound molecule/region of interest). This can betranslated directly to improved contrast to noise (CNR) when using anyof a variety of detection methodologies.

Disclosed herein are surfaces comprising: a) a substrate; b) at leastone hydrophilic polymer coating layer; c) a plurality of oligonucleotidemolecules attached to at least one hydrophilic polymer coating layer;and d) at least one discrete region of the surface that comprises aplurality of clonally-amplified, sample nucleic acid molecules that havebeen annealed to the plurality of attached oligonucleotide molecules,wherein a fluorescence image of the surface exhibits a contrast-to-noiseratio (CNR) of at least 20.

In some embodiments, the fluorescence image of the surface exhibits acontrast-to-noise ratio (CNR) of at least 20 when the sample nucleicacid molecules or complementary sequences thereof are labeled with aCyanine dye-3 (Cy3) fluorophore, and when the fluorescence image isacquired using an Olympus IX83 inverted fluorescence microscope with a20×, 0.75 NA objective, a 532 nm light source, a bandpass and dichroicmirror filter set optimized for 532 nm excitation and Cy3 fluorescenceemission, and a camera (e.g., Andor sCMOS, Zyla 4.2) under non-signalsaturating conditions while the surface is immersed in a buffer (e.g.,25 mM ACES, pH 7.4 buffer).

In some embodiments, the fluorescence image of the surface exhibits acontrast-to-noise ratio (CNR) of at least 40. In some embodiments, thefluorescence image of the surface exhibits a contrast-to-noise ratio(CNR) of at least 60. In some embodiments, the substrate comprisesglass. In some embodiments, the substrate comprises plastic. In someembodiments, the at least one hydrophilic polymer coating layercomprises PEG. In some embodiments, the surface further comprises asecond hydrophilic polymer coating layer. In some embodiments, at leastone hydrophilic polymer layer comprises a branched hydrophilic polymer,e.g., PEG, having at least 4 branches. In some embodiments, at least onehydrophilic polymer layer comprises a branched hydrophilic polymer,e.g., PEG, having at least 8 branches. In some embodiments, at least onehydrophilic polymer layer comprises a branched hydrophilic polymer,e.g., PEG, having at least 16 branches. In some embodiments, at leastone hydrophilic polymer layer comprises a branched hydrophilic polymer,e.g., PEG, having at least 32 branches. In some embodiments, theplurality of oligonucleotide molecules are present at a surface densityof at least 50,000 molecules/μm². In some embodiments, the plurality ofoligonucleotide molecules are present at a surface density of at least100,000 molecules/μm². In some embodiments, the plurality ofoligonucleotide molecules are present at a surface density of at least500,000 molecules/μm². In some embodiments, the sample nucleic acidmolecules were administered at a concentration of no greater than 500 nMprior to annealing and clonal amplification. In some embodiments, thesample nucleic acid molecules were administered at a concentration of nogreater than 20 pM prior to annealing and clonal amplification. In someembodiments, the sample nucleic acid molecules comprise single-strandedmultimeric nucleic acid molecules comprising repeats of a regularlyoccurring monomer unit. In some embodiments, the single-strandedmultimeric nucleic acid molecules are at least 10 kb in length. In someembodiments, the surface further comprises double-stranded monomericcopies of the regularly occurring monomer unit. In some embodiments,said surface is positioned on the interior of a flow channel. In someembodiments, the plurality of oligonucleotide molecules are present at auniform surface density across the surface. In some embodiments, theplurality of oligonucleotide molecules are present at a local surfacedensity of at least 100,000 molecules/μm² at a first position on thesurface, and at a second local surface density at a second position onthe surface. In some embodiments, a background fluorescence intensitymeasured at a region of the surface that is laterally-displaced from theat least one discrete region is no more than 2× of the intensitymeasured at the at least one discrete region prior to said clonalamplification. In some embodiments, the surface comprises a first layercomprising a monolayer of polymer molecules tethered to a surface of thesubstrate; a second layer comprising polymer molecules tethered to thepolymer molecules of the first layer; and a third layer comprisingpolymer molecules tethered to the polymer molecules of the second layer,wherein at least one layer comprises branched polymer molecules. In someembodiments, the third layer further comprises oligonucleotides tetheredto the polymer molecules of the third layer. In some embodiments, theoligonucleotides tethered to the polymer molecules of the third layerare distributed at a plurality of depths throughout the third layer. Insome embodiments, the surface further comprises a fourth layercomprising branched polymer molecules tethered to the polymer moleculesof the third layer, and a fifth layer comprising polymer moleculestethered to the branched polymer molecules of the fourth layer. In someembodiments, the polymer molecules of the fifth layer further compriseoligonucleotides tethered to the polymer molecules of the fifth layer.In some embodiments, the oligonucleotides tethered to the polymermolecules of the fifth layer are distributed at a plurality of depthsthroughout the fifth layer. In some embodiments, the at least onehydrophilic polymer coating layer, comprises a molecule selected fromthe group consisting of polyethylene glycol (PEG), poly(vinyl alcohol)(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylicacid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM),poly(methyl methacrylate) (PMA), poly(-hydroxylethyl methacrylate)(PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate)(POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside,streptavidin, and dextran. In some embodiments, the image of the surfaceexhibits a ratio of fluorescence intensities for specifically-amplified,Cy3-labeled sample nucleic acid molecules, or complementary sequencesthereof, and nonspecific Cy3 dye adsorption background (B_(inter)) of fat least 3:1. In some embodiments, the image of the surface exhibits aratio of fluorescence intensities for specifically-amplified,Cy3-labeled sample nucleic acid molecules, or complementary sequencesthereof, and a combination of nonspecific Cy3 dye adsorption backgroundand nonspecific amplification background (B_(inter)+B_(intra)) of atleast 3:1. In some embodiments, the image of the surface exhibits aratio of fluorescence intensities for specifically-amplified,Cy3-labeled sample nucleic acid molecules, or complementary sequencesthereof, and nonspecific dye adsorption background (B_(inter)) of atleast 5:1. In some embodiments, the image of the surface exhibits aratio of fluorescence intensities for specifically-amplified,Cy3-labeled sample nucleic acid molecules, or complementary sequencesthereof, and a combination of nonspecific Cy3 dye adsorption backgroundand nonspecific amplification background (B_(inter)+B_(intra)) of atleast 5:1.

Also disclosed herein are surfaces comprising: a) a substrate; b) atleast one layers of hydrophilic polymer coating; and c) a plurality ofoligonucleotide molecules attached to at least one of the hydrophilicpolymer coating layers, wherein the surface exhibits a level ofnon-specific Cy3 dye adsorption of less than about 0.25 molecules/μm².

In some embodiments, the surface exhibits a level of non-specific Cy3dye adsorption of less than about 0.1 molecules/μm². In someembodiments, the surface exhibits a ratio of specific Cy3oligonucleotide labeling to non-specific Cy3 dye adsorption is greaterthan about 4:1. In some embodiments, the surface exhibits a ratio ofspecific Cy3 oligonucleotide labeling to non-specific Cy3 dye adsorptionis greater than about 10:1. In some embodiments, the plurality ofoligonucleotide molecules are attached at a surface density of at least10,000 molecules/μm². In some embodiments, the plurality ofoligonucleotide molecules are attached at a surface density of at least100,000 molecules/μm². In some embodiments, the surface furthercomprises a plurality of clonally-amplified clusters of templatemolecules that have been annealed to the plurality of oligonucleotidemolecules, and wherein a fluorescence image of the surface exhibits acontrast-to-noise ratio (CNR) of at least 20. In some embodiments, thecontrast-to-noise ratio (CNR) is at least 50. In some embodiments, thecontrast-to-noise ratio (CNR) is at least 100. In some embodiments, atleast one of the at least two hydrophilic polymer layers comprises abranched polyethylene glycol (PEG) molecule. In some embodiments, thesurface comprises a surface of a capillary lumen or at least oneinternal surface of a flow cell. In some embodiments, the capillarylumen or flow cell is configured for use in performing a nucleic acidhybridization, amplification, or sequencing reaction, or any combinationthereof. In some embodiments, the surface further comprises a branchedpolymer blocking layer. In some embodiments, the branched polymerblocking layer is a branched PEG blocking layer. In some embodiments,the branched polymer blocking layer is covalently tethered to thetopmost hydrophilic polymer layer. In some embodiments, the polymers ofthe first layer comprise primary amine functional groups and thepolymers of the second layer comprise N-hydroxysuccinimide (NHS) esterfunctional groups and, following the deposition of the second layer, thesecond layer is tethered to the first layer using a covalent amidelinkage. In some embodiments, the polymers of the first layer compriseN-hydroxysuccinimide (NHS) ester functional groups and the polymers ofthe second layer comprise primary amine functional groups and, followingthe deposition of the second layer, the second layer is tethered to thefirst layer using a covalent amide linkage. In some embodiments, theoligonucleotides are tethered to the polymer molecules of the second ora third layer at an oligonucleotide-to-polymer molar ratio of about 1:5.In some embodiments, the oligonucleotides are tethered to the polymermolecules of the second or a third layer at anoligonucleotide-to-polymer molar ratio of about 2:5. In someembodiments, the oligonucleotides are tethered to the polymer moleculesof the second or a third layer at an oligonucleotide-to-polymer molarratio of about 3:5. In some embodiments, the oligonucleotides aretethered to the polymer molecules of the second or a third layer at anoligonucleotide-to-polymer molar ratio of about 4:5. In someembodiments, the oligonucleotides are tethered to the polymer moleculesof the second or a third layer at an oligonucleotide-to-polymer molarratio of about 1:1. In some embodiments, the oligonucleotides aretethered to the polymer molecules of the second or a third layer at anoligonucleotide-to-polymer molar ratio of about 4:1. In someembodiments, the oligonucleotides are tethered to the polymer moleculesof the second or a third layer at an oligonucleotide-to-polymer molarratio of about 8:1. In some embodiments, the oligonucleotides aretethered to the polymer molecules of the second or a third layer at anoligonucleotide-to-polymer molar ratio of about 16:1. In someembodiments, the oligonucleotides are tethered to the polymer moleculesof the second or a third layer at an oligonucleotide-to-polymer molarratio of about 32:1. In some embodiments, the oligonucleotides arepresent at a surface density of at least 10,000 molecules per squaremicrometer. In some embodiments, the oligonucleotides are present at asurface density of at least 100,000 molecules per square micrometer. Insome embodiments, the oligonucleotides are uniformly distributed withinthe third layer.

Disclosed herein are methods of depositing oligonucleotides on asubstrate surface, the method comprising: a) conjugating a firsthydrophilic polymer to the substrate surface in a first layer; b)conjugating a second hydrophilic polymer to the first layer to form asecond layer, wherein the hydrophilic polymer molecules of the secondlayer are joined to the first layer by at least two covalent linkagesper molecule; and c) conjugating an outermost hydrophilic polymer to thesecond layer, wherein the outermost hydrophilic polymer moleculescomprise oligonucleotide molecules covalently attached thereto prior toconjugating to the second layer.

In some embodiments, the hydrophilic polymer molecules of the secondlayer are joined to the first layer by at least four covalent linkagesper molecule. In some embodiments, the hydrophilic polymer molecules ofthe second layer are joined to the first layer by at least eightcovalent linkages per molecule. In some embodiments, the method furthercomprises conjugating the outermost hydrophilic polymer directly to thesecond layer. In some embodiments, the method further comprisesconjugating a third hydrophilic polymer to the second layer to form athird layer, and conjugating the outermost hydrophilic polymer to thesecond layer via the third layer. In some embodiments, the methodfurther comprises conjugating a third hydrophilic polymer to the secondlayer to form a third layer, conjugating a fourth hydrophilic polymer tothe third layer to form a fourth layer, and conjugating the outermosthydrophilic polymer to the second layer via the fourth and third layers.In some embodiments, the first hydrophilic polymer comprises PEG. Insome embodiments, the first hydrophilic polymer comprises PGA. In someembodiments, at least one of the hydrophilic polymer layers comprises abranched polymer. In some embodiments, the branched polymer comprises atleast 4 branches. In some embodiments, the branched polymer comprises atleast 8 branches. In some embodiments, the branched polymer comprises 16to 32 branches. In some embodiments, the hydrophilic polymer moleculesof the fourth layer are joined to the third layer by at least twocovalent linkages per molecule. In some embodiments, the hydrophilicpolymer molecules of the fourth layer are joined to the third layer byat least four covalent linkages per molecule. In some embodiments, thehydrophilic polymer molecules of the fourth layer are joined to thethird layer by at least eight covalent linkages per molecule. In someembodiments, the hydrophilic polymer is delivered to the substratesurface in a solvent comprising ethanol. In some embodiments, thehydrophilic polymer is delivered to the substrate surface in a solventcomprising methanol. In some embodiments, the hydrophilic polymer isdelivered to the substrate surface in a solvent comprising dimethylsulfoxide (DMSO). In some embodiments, the hydrophilic polymer isdelivered to the substrate surface in a solvent comprising acetonitrile.In some embodiments, the hydrophilic polymer is delivered to thesubstrate surface in a solvent comprising buffered phosphate. In someembodiments, the hydrophilic polymer is delivered to the substratesurface in a solvent comprising buffered 3-(N-morpholino)propanesulfonicacid (MOPS). In some embodiments, the hydrophilic polymer is deliveredto the substrate surface in a solvent comprising 75% acetonitrile, 25%phosphate buffer. In some embodiments, the hydrophilic polymer isdelivered to the substrate surface in a solvent comprising 90% methanol,10% MOPS buffer.

Disclosed herein are surfaces comprising oligonucleotides at a densityof at least 10,000 molecules per square micrometer, wherein theoligonucleotides are tethered to the surface via a multilayeredhydrophilic polymeric stratum, and wherein the oligonucleotides areevenly distributed throughout an outermost layer of the multilayeredhydrophilic polymeric stratum.

In some embodiments, the oligonucleotides are distributed at a surfacedensity of at least 50,000 molecules per square micrometer. In someembodiments, the oligonucleotides are distributed at a surface densityof at least 100,000 molecules per square micrometer. In someembodiments, the oligonucleotides are distributed at a surface densityof at least 500,000 molecules per square micrometer. In someembodiments, at least 10% of the tethered oligonucleotides are annealedto target (or sample) oligonucleotides. In some embodiments, themultilayered hydrophilic polymeric stratum is saturated by a hydrophilicsolvent. In some embodiments, the surface comprises a surface of aglass, fused-silica, silicon, or polymer (e.g., plastic) substrate. Insome embodiments, the multilayered hydrophilic polymeric stratumcomprises three or more polymer layers. In some embodiments, themultilayered hydrophilic polymeric stratum comprises five or morepolymer layers. In some embodiments, one or more layers of thehydrophilic polymeric stratum comprise branched PEG, branched PVA,branched poly(vinyl pyridine), branched PVP, branched PAA, branchedPNIPAM, branched PMA, branched PHEMA, branched PEGMA, branched PGA,branched poly-lysine, branched poly-glucoside, or dextran. In someembodiments, one or more layers of the hydrophilic polymeric stratumcomprise branched PEG molecules. In some embodiments, the branched PEGmolecules comprises at least 4 branches. In some embodiments, thebranched PEG molecules comprise at least 8 branches. In someembodiments, the branched PEG molecules comprise 16 to 32 branches. Insome embodiments, at least a first layer and a second layer of thehydrophilic polymeric stratum are tethered to each other using acovalent amide linkage. In some embodiments, at least a first layer anda second layer of the hydrophilic polymeric stratum are tethered to eachother by at least two covalent linkages per polymer molecule. In someembodiments, at least a first layer and a second layer of thehydrophilic polymeric stratum are tethered to each other by at leastfour covalent linkages per polymer molecule. In some embodiments, atleast a first layer and a second layer of the hydrophilic polymericstratum are tethered to each other by at least eight covalent linkagesper polymer molecule. In some embodiments, the surface density oftethered oligonucleotides is at least 50,000 molecules per squaremicrometer. In some embodiments, the surface density of tetheredoligonucleotides is at least 100,000 molecules per square micrometer. Insome embodiments, the surface exhibits nonspecific binding of CY3 dye ofless than 0.25 molecules/μm². In some embodiments, the surface furthercomprises clusters of clonally-amplified copies of the annealed targetoligonucleotides, wherein substantially all of the clonally-amplifiedcopies of the annealed target oligonucleotides comprise a Cy3-labelednucleotide annealed at a first position, and wherein a fluorescenceimage of the surface exhibits a contrast-to-noise (CNR) ratio of atleast 20. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 50. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 100. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 150. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 200. In some embodiments, the clonally-amplified copies of theannealed target oligonucleotides are prepared using a bridgeamplification protocol. In some embodiments, the clonally-amplifiedcopies of the annealed target oligonucleotides are prepared using anisothermal bridge amplification protocol. In some embodiments, theclonally-amplified copies of the annealed target oligonucleotides areprepared using a rolling circle amplification (RCA) protocol. In someembodiments, the clonally-amplified copies of the annealed targetoligonucleotides are prepared using a helicase-dependent amplificationprotocol. In some embodiments, the clonally-amplified copies of theannealed target oligonucleotides are prepared using arecombinase-dependent amplification protocol. In some embodiments, theclonally-amplified copies of the annealed target oligonucleotides areprepared using a single-stranded binding (SSB) protein-dependentamplification protocol. In some embodiments, the surface comprises asurface of a capillary lumen or at least one internal surface of a flowcell. In some embodiments, the capillary lumen or flow cell isconfigured for use in performing a nucleic acid hybridization,amplification, or sequencing reaction, or any combination thereof.

Disclosed herein are methods for performing solid-phase nucleic acidhybridization, the method comprising: a) providing any one of thesurfaces disclosed herein; and b) performing a solid-phase nucleic acidhybridization reaction, wherein template nucleic acid molecules areannealed to the tethered oligonucleotides. Also disclosed herein aremethods for performing solid-phase nucleic acid amplification, themethod comprising: a) providing any one of the surfaces disclosedherein; and b) performing a solid-phase nucleic acid amplificationreaction using template nucleic acid molecules hybridized to thetethered oligonucleotides.

In some embodiments, the solid phase nucleic acid amplificationcomprises thermocycling. In some embodiments, the solid phase nucleicacid amplification comprises isothermal amplification. In someembodiments, the solid phase nucleic acid amplification comprisesrolling circle amplification. In some embodiments, the solid phasenucleic acid amplification comprises bridge amplification. In someembodiments, the solid phase nucleic acid amplification comprisesisothermal bridge amplification. In some embodiments, the solid phasenucleic acid amplification comprises multiple displacementamplification. In some embodiments, the solid phase nucleic acidamplification comprises helicase treatment. In some embodiments, thesolid phase nucleic acid amplification comprises recombinase treatment.In some embodiments, there is no change in a surface density of tetheredoligonucleotides over at least 30 cycles of the solid-phase nucleic acidamplification reaction. In some embodiments, there is no change in asurface density of tethered oligonucleotides over at least 40 cycles ofthe solid-phase nucleic acid amplification reaction. In someembodiments, there is no change in a surface density of tetheredoligonucleotides over at least 50 cycles of the solid-phase nucleic acidamplification reaction.

Disclosed herein are methods of performing nucleic acid sequencedetermination, the method comprising: a) providing any one of thesurfaces disclosed herein; b) performing a solid-phase nucleic acidamplification reaction using template nucleic acid molecules hybridizedto the tethered oligonucleotides; and c) performing a cyclic series ofsingle nucleotide binding or incorporation reactions, wherein thenucleotides are labeled with a detectable tag.

In some embodiments, the detectable tag is a fluorophore. In someembodiments, the fluorophore is Cy3, and wherein a fluorescence image ofthe surface acquired as described elsewhere herein under non-signalsaturating conditions after the binding or incorporation of a firstCy3-labeled nucleotide exhibits a contrast-to-noise (CNR) ratio of atleast 20. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 50. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 100. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 150. In some embodiments, the contrast-to-noise ratio (CNR) is atleast 200. In some embodiments, the solid-phase nucleic acidamplification reaction comprises a bridge amplification reaction. Insome embodiments, the solid-phase nucleic acid amplification reactioncomprises an isothermal bridge amplification reaction. In someembodiments, the solid-phase nucleic acid amplification reactioncomprises a rolling circle amplification (RCA) reaction. In someembodiments, the solid-phase nucleic acid amplification reactioncomprises a helicase-dependent amplification reaction. In someembodiments, the solid-phase nucleic acid amplification reactioncomprises a recombinase-dependent amplification reaction.

Disclosed herein are devices for performing nucleic acid amplification,the device comprising: a) any one of the surfaces disclosed herein;wherein the surface comprises a surface of a capillary lumen or at leastone internal surface of a flow cell.

In some embodiments, the device further comprises at least one fluidinlet into the capillary lumen. In some embodiments, the device furthercomprises at least one fluid outlet. In some embodiments, the devicefurther comprises at least one pump. In some embodiments, the devicefurther comprises at least one fluid mixing manifold. In someembodiments, the device further comprises at least one temperaturecontrol element. In some embodiment, the device further comprises atleast one optical window.

Disclosed here are systems for performing nucleic acid sequencing, thesystem comprising: a) at least one of the devices disclosed herein; b) afluid control module; and c) an imaging module.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a schematic illustration of one embodiment of the lowbinding solid supports of the present disclosure in which the supportcomprises a glass substrate and alternating layers of hydrophiliccoatings which are covalently or non-covalently adhered to the glass,and which further comprises chemically-reactive functional groups thatserve as attachment sites for oligonucleotide primers.

FIG. 2 provides a schematic illustration of the use of a silane reactionto covalently couple a first polymer to a substrate (e.g., glass)surface to create a first polymer layer.

FIG. 3 provides a schematic illustration of the covalent coupling of abranched polymer to a surface such as that illustrated in FIG. 2 to forma second polymer layer on a substrate surface.

FIG. 4 provides a schematic illustration of a coupling reaction used tocovalently attach one or more oligonucleotide adapter or primersequences (e.g., sequence 1 (dotted line) and sequence 2 (dashed line))to a branched polymer.

FIG. 5 provides a schematic illustration of the covalent coupling of abranched polymer comprising covalently-attached oligonucleotide adapteror primer sequences to a surface such as that illustrated in FIG. 3 toform a third polymer layer on a substrate surface.

FIGS. 6A-B provide examples of simulated fluorescence intensity datathat illustrate the difference between using signal-to-noise ratio (SNR)and contrast-to-noise ratio (CNR) as metrics of data quality in nucleicacid sequencing and base-calling applications. FIG. 6A: example ofsimulated data for which the SNR=2 and CNR=1.25. FIG. 6B: example ofsimulated data for which SNR=2 and CNR=12.29.

FIG. 7 provides an example of how improved CNR impacts the imaging timesrequired for accurate detection of and signal classification(base-calling) for clonally-amplified nucleic acid colonies on a solidsupport.

FIG. 8 illustrates the different images acquired for different cycles ofa nucleic acid sequencing reaction performed on a solid support due tothe different labeled nucleotides incorporated into the complementarystrands for each clonally-amplified template molecule. The figure alsoillustrates the different background contributions to the overalldetection signal for detection platforms that require iterative spotdetection through distinguishing nucleotide-specific signal from noisyinterstitial background and intrastitial background images.

FIG. 9 provides an example of image data from a study to determine therelative levels of non-specific binding of a green fluorescent dye toglass substrate surfaces treated according to different surfacemodification protocols.

FIG. 10 provides an example of image data from a study to determine therelative levels of non-specific binding of a red fluorescent dye toglass substrate surfaces treated according to different surfacemodification protocols.

FIG. 11 provides an example of oligonucleotide primer grafting data forsubstrate surfaces treated according to different surface modificationprotocols.

FIG. 12 provides examples of replicate images acquired during anon-specific binding test of substrate surfaces treated according todifferent surface modification protocols.

FIG. 13 provides an example of data for non-specific binding of asequencing dye mixture to substrate surfaces treated according todifferent surface modification protocols. For comparison purposes, thefluorescence intensity measured under the same set of experimentalconditions for non-specific binding of the sequencing dye mixture to asingle bead grafted with Cy3 labeled oligonucleotides is about 1,500counts.

FIG. 14 provides an example of images and data for non-specific bindingof green and red fluorescent dyes to substrate surfaces treatedaccording to different surface modification protocols. For comparisonpurposes, the fluorescence intensity of a clonally-amplified templatecolony measured under the same set of experimental conditions aftercoupling a single Cy3-labeled nucleotide base is about 1,500 counts.

FIG. 15 provides an example of images and data demonstrating “tunable”nucleic acid amplification on a low binding solid support by varying theoligonucleotide primer density on the substrate. Blue histogram: lowprimer density. Red histogram: high primer density. The combination oflow non-specific binding and tunable nucleic acid amplificationefficiency through adjustment of oligonucleotide primer density yieldshigh CNRs and subsequent improvements in nucleic acid sequencingperformance.

FIG. 16 provides examples of fluorescence images of the low bindingsolid supports of the present disclosure on which tetheredoligonucleotides have been amplified using different primer densities,isothermal amplification methods, and amplification buffer additives.

FIG. 17 provides examples of gel images that demonstrate reduction ofnon-specific nucleic acid amplification through the use of amplificationbuffer additives while maintaining specific amplification of the targetsequence. The gel images reveal a band corresponding to specificamplification of the target (arrow) and other gel quantifiedamplification products.

FIG. 18 provides examples of fluorescent images that demonstrate theimpact of formulation changes to improve amplification specificity on alow binding support surface.

FIGS. 19A-B provide non-limiting examples of image data that demonstratethe improvements in hybridization stringency, speed, and efficacy thatmay be achieved through the reformulation of the hybridization bufferused for solid-phase nucleic acid amplification, as described herein.FIG. 19A provides examples of image data for two different hybridizationbuffer formulations and protocols. FIG. 19B provides an example of thecorresponding image data obtained using a standard hybridization bufferand protocol.

FIG. 20 illustrates a workflow for nucleic acid sequencing using thedisclosed low binding supports and amplification reaction formulationsof the present disclosure, and non-limiting examples of the processingtimes that may be achieved.

FIG. 21 provides an example of fluorescence image and intensity data fora low-binding support of the present disclosure on which solid-phasenucleic acid amplification was performed to create clonally-amplifiedclusters of a template oligonucleotide sequence.

FIG. 22 provides a second example of fluorescence image and intensitydata for a low-binding support of the present disclosure on whichsolid-phase nucleic acid amplification was performed to createclonally-amplified clusters of a template oligonucleotide sequence.

FIG. 23 provides an example of fluorescence image and intensity data fora low-binding support of the present disclosure on which solid-phasenucleic acid amplification was performed to create clonally-amplifiedclusters of a template oligonucleotide sequence.

FIG. 24 provides an example of a fluorescence calibration curve used toestimate the surface density of primer oligonucleotides tethered to asupport surface.

FIGS. 25A-B provide non-limiting examples of modified glass and polymersurfaces of the present disclosure having bound amplicons comprisingfluorescently labeled nucleotides. FIG. 25A: modified glass surface. Atinset, one sees that the surface yields a CNR of 226. FIG. 25B: modifiedplastic surface. At inset, one sees that the surface yields a CNR of109.

FIGS. 26A-B provide analysis of the images at FIG. 25A and FIG. 25B. AtFIG. 26A, one sees signal intensity, left, and background intensity,right, for each of glass and plastic surfaces. For each, signalintensity is substantially greater than background intensity. At FIG.26B, one sees a graphic depiction of CNR values for each of glass andplastic surfaces. At left, glass yields a CNR of 226, while at right,plastic yields a CNR of 109, consistent with the insets of FIG. 25A andFIG. 25B.

FIG. 27 provides analysis of surfaces as to the accuracy of their data.Data is collected in two channels and is depicted in scatter pot from(top) and quantified (bottom) for each of a commercially available, lowCNR and high CNR surface from left to right.

FIG. 28 provides a schematic illustration of a multimeric targetoligonucleotide sequence hybridized to a surface comprising a highsurface density of oligonucleotide adapter or primer molecules (left)and to a surface comprising a lower surface density of oligonucleotideadapter or primer molecules (right).

FIG. 29 provides a comparison of the experimental outcomes forperforming a traditional hybridization reaction on a low-binding supportsurface of the present disclosure and performing an optimizedhybridization reaction on the low-binding support surface of the presentdisclosure.

FIG. 30 provides an illustration of the experimental outcome ofperforming a traditional hybridization reaction on a low-binding supportof the present disclosure, followed by performing RCA or Bridgeamplification.

FIG. 31 provides an illustration of the experimental outcome ofperforming an optimized hybridization reaction on a low-binding supportof the present disclosure, followed by performing RCA or Bridgeamplification.

FIG. 32 provides an illustration of the experimental outcome ofperforming an optimized hybridization reaction on a low-binding supportof the present disclosure prepared using improved coupling chemistry forattaching oligonucleotide adaptor or primer molecules to the surface,followed by performing RCA or Bridge amplification.

FIG. 33 provides non-limiting examples of fluorescence images of atraditional support surface and a low-binding support surface of thepresent disclosure to which target oligonucleotides have been hybridizedand amplified. The plot illustrates the contrast-to-noise ratiosmeasured from images such as those provided in the examples for apolyacrylamide surface with bridge amplification protocol, low bindingsupport with a standard bridge amplification protocol and a primerdensity<1000 oligonucleotides/um2, and a low-binding support surface ofthe present disclosure in combination with an improved solid-phasenucleic acid amplification method on a surface with a primerdensity>1000 oligonucleotides/um2.

DETAILED DESCRIPTION

Disclosed herein are novel solid supports for use in solid-phase nucleicacid amplification and sequencing, or other bioassay applications. Thesolid supports disclosed herein exhibit low non-specific binding ofproteins and other amplification reaction components, and improvedstability to repetitive exposure to different solvents, changes intemperature, chemical affronts such as low pH, or long term storage.

Alone or in combination with improved nucleic acid hybridization andamplification protocols, some supports disclosed herein lead to one ormore of: (i) reduced requirements for the amount of starting materialnecessary, (ii) lowered temperature requirements for isothermal orthermal ramping amplification protocols, (iii) increased amplificationrates, (iv) increased amplification specificity (that is, more selectiveamplification of the single-stranded template molecules of the amplifiedcolonies while decreasing non-specific amplification of surface primersand primer-dimers), and (v) allow greater discrimination ofsequence-specific signal from background signals (such as signalsarising from both interstitial and intrastitial background), therebyproviding improved contrast-to-noise ratio (CNR) and base-callingaccuracy compared to conventional nucleic acid amplification andsequencing methodologies.

The starting point for achieving the aforementioned improvements, or anycombination thereof, are the disclosed low non-specific binding supportscomprising one or more polymer coatings, e.g., PEG polymer films, thatminimize non-specific binding of protein and labeled nucleotides to thesolid support. The subsequent demonstration of improved nucleic acidhybridization and amplification rates and specificity may be achievedthrough one or more of the following additional aspects of the presentdisclosure: (i) primer design (sequence and/or modifications), (ii)control of tethered primer density on the solid support, (iii) thesurface composition of the solid support, (iv) the surface polymerdensity of the solid support, (v) the use of improved hybridizationconditions before and during amplification, and/or (vi) the use ofimproved amplification formulations that decrease non-specific primeramplification or increase template amplification efficiency.

The advantages of the disclosed low non-specific binding supports andassociated hybridization and amplification methods confer one or more ofthe following additional advantages for any sequencing system: (i)decreased fluidic wash times (due to reduced non-specific binding, andthus faster sequencing cycle times), (ii) decreased imaging times (andthus faster turnaround times for assay readout and sequencing cycles),(iii) decreased overall work flow time requirements (due to decreasedcycle times), (iv) decreased detection instrumentation costs (due to theimprovements in CNR), (v) improved readout (base-calling) accuracy (dueto improvements in CNR), (vi) improved reagent stability and decreasedreagent usage requirements (and thus reduced reagents costs), and (vii)fewer run-time failures due to nucleic acid amplification failures.

The low binding hydrophilic surfaces (multilayer and/or monolayer) forsurface bioassays, e.g., genotyping and sequencing assays, are createdby using any combination of the following.

Polar protic, polar aprotic and/or nonpolar solvents for depositingand/or coupling linear or multi-branched hydrophilic polymer subunits ona substrate surface. Some multi-branched hydrophilic polymer subunitsmay contain functional end groups to promote covalent coupling ornon-covalent binding interactions with other polymer subunits. Examplesof suitable functional end groups include biotin, methoxy ether,carboxylate, amine, ester compounds, azide, alkyne, maleimide, thiol,and silane groups.

Any combination of linear, branched, or multi-branched polymer subunitscoupled through subsequent layered addition via modified couplingchemistry/solvent/buffering systems that may include individual subunitswith orthogonal end coupling chemistries or any of the respectivecombinations, such that resultant surface is hydrophilic and exhibitslow nonspecific binding of proteins and other molecular assaycomponents. In some instances, the hydrophilic, functionalized substratesurfaces of the present disclosure exhibit contact angle measurementsthat do not exceed 35 degrees.

Compatible buffering systems in addition to the aforementioned solvents,with a desirable pH range of 5-10. Examples include, but are not limitedto, phosphate buffered saline, phosphate buffer, TAPS, MES, MOPS, or anycombination of these.

Subsequent biomolecule attachment (e.g., of proteins, peptides, nucleicacids, oligonucleotides, or cells) on the low binding/hydrophilicsubstrates via any of a variety of individual conjugation chemistries tobe described below, or any combination thereof. Layer deposition and/orconjugation reactions may be performed using solvent mixtures which maycontain any ratio of the following components: ethanol, methanol,acetonitrile, acetone, DMSO, DMF, H₂O, and the like. In addition,compatible buffering systems in the desirable pH range of 5-10 may beused for controlling the rate and efficiency of deposition and coupling,whereby coupling rates is excess of >5× of those for conventionalaqueous buffer-based methods may be achieved.

Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art inthe field to which this disclosure belongs.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated.

As used herein, the term ‘about’ a number refers to that number plus orminus 10% of that number. The term ‘about’ when used in the context of arange refers to that range minus 10% of its lowest value and plus 10% ofits greatest value.

As used herein, the phrase ‘at least one of’ in the context of a seriesencompasses lists including a single member of the series, two membersof the series, up to and including all members of the series, alone orin some cases in combination with unlisted components.

As used herein, fluorescence is ‘specific’ if it arises fromfluorophores that are annealed or otherwise tethered to the surface,such as through a nucleic acid having a region of reversecomplementarity to a corresponding segment of an oligo on the surfaceand annealed to said corresponding segment. This fluorescence iscontrasted with fluorescence arising from fluorophores not tethered tothe surface through such an annealing process, or in some cases tobackground florescence of the surface.

Nucleic acids: As used herein, a “nucleic acid” (also referred to as a“polynucleotide”, “oligonucleotide”, ribonucleic acid (RNA), ordeoxyribonucleic acid (DNA)) is a linear polymer of two or morenucleotides joined by covalent internucleosidic linkages, or variants orfunctional fragments thereof. In naturally occurring examples of nucleicacids, the internucleoside linkage is typically a phosphodiester bond.However, other examples optionally comprise other internucleosidelinkages, such as phosphorothiolate linkages and may or may not comprisea phosphate group. Nucleic acids include double- and single-strandedDNA, as well as double- and single-stranded RNA, DNA/RNA hybrids,peptide-nucleic acids (PNAs), hybrids between PNAs and DNA or RNA, andmay also include other types of nucleic acid modifications.

As used herein, a “nucleotide” refers to a nucleotide, nucleoside, oranalog thereof. In some cases, the nucleotide is an N- or C-glycoside ofa purine or pyrimidine base (e.g., a deoxyribonucleoside containing2-deoxy-D-ribose or ribonucleoside containing D-ribose). Examples ofother nucleotide analogs include, but are not limited to,phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, and the like.

Nucleic acids may optionally be attached to one or more non-nucleotidemoieties such as labels and other small molecules, large molecules (suchas proteins, lipids, sugars, etc.), and solid or semi-solid supports,for example through covalent or non-covalent linkages with either the 5′or 3′ end of the nucleic acid. Labels include any moiety that isdetectable using any of a variety of detection methods known to those ofskill in the art, and thus renders the attached oligonucleotide ornucleic acid similarly detectable. Some labels emit electromagneticradiation that is optically detectable or visible. Alternately or incombination, some labels comprise a mass tag that renders the labeledoligonucleotide or nucleic acid visible in mass spectral data, or aredox tag that renders the labeled oligonucleotide or nucleic aciddetectable by amperometry or voltammetry. Some labels comprise amagnetic tag that facilitates separation and/or purification of thelabeled oligonucleotide or nucleic acid. The nucleotide orpolynucleotide is often not attached to a label, and the presence of theoligonucleotide or nucleic acid is directly detected.

The disclosed low non-specific binding supports and associated nucleicacid hybridization and amplification methods may be used for theanalysis of nucleic acid molecules derived from any of a variety ofdifferent cell, tissue, or sample types known to those of skill in theart. For example, nucleic acids may be extracted from cells, or tissuesamples comprising one or more types of cells, derived from eukaryotes(such as animals, plants, fungi, protista), archaebacteria, oreubacteria. In some cases, nucleic acids may be extracted fromprokaryotic or eukaryotic cells, such as adherent or non-adherenteukaryotic cells. Nucleic acids are variously extracted from, forexample, primary or immortalized rodent, porcine, feline, canine,bovine, equine, primate, or human cell lines. Nucleic acids may beextracted from any of a variety of different cell, organ, or tissuetypes (e.g., white blood cells, red blood cells, platelets, epithelialcells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts,skeletal muscle cells, smooth muscle cells, gametes, or cells from theheart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder,stomach, colon, or small intestine). Nucleic acids may be extracted fromnormal or healthy cells. Alternately or in combination, acids areextracted from diseased cells, such as cancerous cells, or frompathogenic cells that are infecting a host. Some nucleic acids may beextracted from a distinct subset of cell types, e.g., immune cells (suchas T cells, cytotoxic (killer) T cells, helper T cells, alpha beta Tcells, gamma delta T cells, T cell progenitors, B cells, B-cellprogenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes,granulocytes, Natural Killer cells, plasma cells, memory cells,neutrophils, eosinophils, basophils, mast cells, monocytes, dendriticcells, and/or macrophages, or any combination thereof), undifferentiatedhuman stem cells, human stem cells that have been induced todifferentiate, rare cells (e.g., circulating tumor cells (CTCs),circulating epithelial cells, circulating endothelial cells, circulatingendometrial cells, bone marrow cells, progenitor cells, foam cells,mesenchymal cells, or trophoblasts). Other cells are contemplated andconsistent with the disclosure herein.

Nucleic acid extraction from cells or other biological samples may beperformed using any of a number of techniques known to those of skill inthe art. For example, a typical DNA extraction procedure comprises (i)collection of the cell sample or tissue sample from which DNA is to beextracted, (ii) disruption of cell membranes (i.e., cell lysis) torelease DNA and other cytoplasmic components, (iii) treatment of thelysed sample with a concentrated salt solution to precipitate proteins,lipids, and RNA, followed by centrifugation to separate out theprecipitated proteins, lipids, and RNA, and (iv) purification of DNAfrom the supernatant to remove detergents, proteins, salts, or otherreagents used during the cell membrane lysis step.

A variety of suitable commercial nucleic acid extraction andpurification kits are consistent with the disclosure herein. Examplesinclude, but are not limited to, the QIAamp® kits (for isolation ofgenomic DNA from human samples) and DNAeasy kits (for isolation ofgenomic DNA from animal or plant samples) from Qiagen (Germantown, Md.),or the Maxwell® and ReliaPrep™ series of kits from Promega™ (Madison,Wis.).

Low non-specific binding supports for solid-phase nucleic acidhybridization and amplification: Disclosed herein are solid supportscomprising low non-specific binding surface compositions that enableimproved nucleic acid hybridization and amplification performance. Ingeneral, the disclosed supports may comprise a substrate (or supportstructure), one or more layers of a covalently or non-covalentlyattached low-binding, chemical modification layers, e.g., silane layers,polymer films, and one or more covalently or non-covalently attachedprimer sequences that may be used for tethering single-stranded templateoligonucleotides to the support surface (FIG. 1). In some instances, theformulation of the surface, e.g., the chemical composition of one ormore layers, the coupling chemistry used to cross-link the one or morelayers to the support surface and/or to each other, and the total numberof layers, may be varied such that non-specific binding of proteins,nucleic acid molecules, and other hybridization and amplificationreaction components to the support surface is minimized or reducedrelative to a comparable monolayer. Often, the formulation of thesurface may be varied such that non-specific hybridization on thesupport surface is minimized or reduced relative to a comparablemonolayer. The formulation of the surface may be varied such thatnon-specific amplification on the support surface is minimized orreduced relative to a comparable monolayer. The formulation of thesurface may be varied such that specific amplification rates and/oryields on the support surface are maximized. Amplification levelssuitable for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, or more than 30 amplification cycles in somecases disclosed herein.

Examples of materials from which the substrate or support structure maybe fabricated include, but are not limited to, glass, fused-silica,silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof. Various compositionsof both glass and plastic substrates are contemplated.

The substrate or support structure may be rendered in any of a varietyof geometries and dimensions known to those of skill in the art, and maycomprise any of a variety of materials known to those of skill in theart. For example, in some instances the substrate or support structuremay be locally planar (e.g., comprising a microscope slide or thesurface of a microscope slide). Globally, the substrate or supportstructure may be cylindrical (e.g., comprising a capillary or theinterior surface of a capillary), spherical (e.g., comprising the outersurface of a non-porous bead), or irregular (e.g., comprising the outersurface of an irregularly-shaped, non-porous bead or particle). In someinstances, the surface of the substrate or support structure used fornucleic acid hybridization and amplification may be a solid, non-poroussurface. In some instances, the surface of the substrate or supportstructure used for nucleic acid hybridization and amplification may beporous, such that the coatings described herein penetrate the poroussurface, and nucleic acid hybridization and amplification reactionsperformed thereon may occur within the pores.

The substrate or support structure that comprises the one or morechemically-modified layers, e.g., layers of a low non-specific bindingpolymer, may be independent or integrated into another structure orassembly. For example, in some instances, the substrate or supportstructure may comprise one or more surfaces within an integrated orassembled microfluidic flow cell. The substrate or support structure maycomprise one or more surfaces within a microplate format, e.g., thebottom surface of the wells in a microplate. As noted above, in somepreferred embodiments, the substrate or support structure comprises theinterior surface (such as the lumen surface) of a capillary. Inalternate preferred embodiments the substrate or support structurecomprises the interior surface (such as the lumen surface) of acapillary etched into a planar chip.

The chemical modification layers may be applied uniformly across thesurface of the substrate or support structure. Alternately, the surfaceof the substrate or support structure may be non-uniformly distributedor patterned, such that the chemical modification layers are confined toone or more discrete regions of the substrate. For example, thesubstrate surface may be patterned using photolithographic techniques tocreate an ordered array or random pattern of chemically-modified regionson the surface. Alternately or in combination, the substrate surface maybe patterned using, e.g., contact printing and/or ink-jet printingtechniques. In some instances, an ordered array or random pattern ofchemically-modified discrete regions may comprise at least 1, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or morediscrete regions, or any intermediate number spanned by the rangeherein.

In order to achieve low nonspecific binding surfaces (also referred toherein as “low binding” or “passivated” surfaces), hydrophilic polymersmay be nonspecifically adsorbed or covalently grafted to the substrateor support surface. Typically, passivation is performed utilizingpoly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) orpolyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilicpolymers with different molecular weights and end groups that are linkedto a surface using, for example, silane chemistry. The end groups distalfrom the surface can include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In someinstances, two or more layers of a hydrophilic polymer, e.g., a linearpolymer, branched polymer, or multi-branched polymer, may be depositedon the surface. In some instances, two or more layers may be covalentlycoupled to each other or internally cross-linked to improve thestability of the resulting surface. In some instances, oligonucleotideprimers with different base sequences and base modifications (or otherbiomolecules, e.g., enzymes or antibodies) may be tethered to theresulting surface layer at various surface densities. In some instances,for example, both surface functional group density and oligonucleotideconcentration may be varied to target a certain primer density range.Additionally, primer density can be controlled by dilutingoligonucleotide with other molecules that carry the same functionalgroup. For example, amine-labeled oligonucleotide can be diluted withamine-labeled polyethylene glycol in a reaction with an NHS-ester coatedsurface to reduce the final primer density. Primers with differentlengths of linker between the hybridization region and the surfaceattachment functional group can also be applied to control surfacedensity. Example of suitable linkers include poly-T and poly-A strandsat the 5′ end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3to 20 monomer units), and carbon-chain (e.g., C6, C12, C18, etc.). Tomeasure the primer density, fluorescently-labeled primers may betethered to the surface and a fluorescence reading then compared withthat for a dye solution of known concentration.

In some embodiments, the hydrophilic polymer can be a cross linkedpolymer. In some embodiments, the cross-linked polymer can include onetype of polymer cross linked with another type of polymer. Examples ofthe crossed-linked polymer can include poly(ethylene glycol)cross-linked with another polymer selected from polyethylene oxide (PEO)or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or other hydrophilicpolymers. In some embodiments, the cross-linked polymer can be apoly(ethylene glycol) cross-linked with polyacrylamide.

As a result of the surface passivation techniques disclosed herein,proteins, nucleic acids, and other biomolecules do not “stick” to thesubstrates, that is, they exhibit low nonspecific binding (NSB).Examples are shown below using standard monolayer surface preparationswith varying glass preparation conditions. Hydrophilic surface that havebeen passivated to achieve ultra-low NSB for proteins and nucleic acidsrequire novel reaction conditions to improve primer deposition reactionefficiencies, hybridization performance, and induce effectiveamplification. All of these processes require oligonucleotide attachmentand subsequent protein binding and delivery to a low binding surface. Asdescribed below, the combination of a new primer surface conjugationformulation (Cy3 oligonucleotide graft titration) and resultingultra-low non-specific background (NSB functional tests performed usingred and green fluorescent dyes) yielded results that demonstrate theviability of the disclosed approaches. Some surfaces disclosed hereinexhibit a ratio of specific (e.g., hybridization to a tethered primer orprobe) to nonspecific binding (e.g., B_(inter)) of a fluorophore such asCy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediatevalue spanned by the range herein. Some surfaces disclosed hereinexhibit a ratio of specific to nonspecific fluorescence signal (e.g.,for specifically-hybridized to nonspecifically bound labeledoligonucleotides, or for specifically-amplified to nonspecifically-bound(B_(inter)) or non-specifically amplified (B_(intra)) labeledoligonucleotides or a combination thereof (B_(inter)+B_(intra))) for afluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or anyintermediate value spanned by the range herein.

In order to scale primer surface density and add additionaldimensionality to hydrophilic or amphoteric surfaces, substratescomprising multi-layer coatings of PEG and other hydrophilic polymershave been developed. By using hydrophilic and amphoteric surfacelayering approaches that include, but are not limited to, thepolymer/co-polymer materials described below, it is possible to increaseprimer loading density on the surface significantly. Traditional PEGcoating approaches use monolayer primer deposition, which have beengenerally reported for single molecule applications, but do not yieldhigh copy numbers for nucleic acid amplification applications. Asdescribed herein “layering” can be accomplished using traditionalcrosslinking approaches with any compatible polymer or monomer subunitssuch that a surface comprising two or more highly crosslinked layers canbe built sequentially. Examples of suitable polymers include, but arenot limited to, streptavidin, poly acrylamide, polyester, dextran,poly-lysine, and copolymers of poly-lysine and PEG. In some instances,the different layers may be attached to each other through any of avariety of conjugation reactions including, but not limited to,biotin-streptavidin binding, azide-alkyne click reaction, amine-NHSester reaction, thiol-maleimide reaction, and ionic interactions betweenpositively charged polymer and negatively charged polymer. In someinstances, high primer density materials may be constructed in solutionand subsequently layered onto the surface in multiple steps.

FIG. 2 provides a schematic illustration of one non-limiting example ofgrafting a first hydrophilic polymer layer to a substrate, e.g., a glasssubstrate. Following cleaning of the glass surface using any of avariety of methods known to those of skill in the art (e.g., treatmentwith a Piranha solution, plasma cleaning, etc.), the substrate istreated with a silane solution (e.g., a silane PEG 5K solution), rinsed,dried, and cured at an elevated temperature to form a covalent bond withsurface. The end of the polymer distal from the surface may comprise anyof a variety of chemically-reactive functional groups or protectedfunctional groups. An amine-reactive NHS group is illustrated in FIG. 2.

FIG. 3 provides a schematic illustration of one non-limiting example ofcoupling a derivatized substrate comprising a first polymer layer havingan NHS-functional group such as that illustrated in FIG. 2 with aprimary amine-functionalized branched polymer (e.g., a 16-branch or32-branch PEG polymer (also referred to as 16-arm or 32-arm PEG,respectively)) to create a second hydrophilic polymer layer comprisingan excess of unreacted functional groups.

FIG. 4 provides a schematic illustration of one non-limiting example ofreacting a branched polymer comprising reactive functional groups (e.g.,a 4-branched NHS-PEG) with one or more oligonucleotide adapter or primersequences in solution (e.g., oligonucleotides comprising a primaryamine, as illustrated by the dotted and dashed lines) prior todepositing on a substrate surface to create a hydrophilic layercomprising covalently-attached oligonucleotide molecules. By varying themolar ratio of the oligonucleotide molecule(s) (or other biomolecules tobe tethered, e.g., peptides, proteins, enzymes, antibodies, etc.) tothat of the branched polymer, one may vary the resulting surface densityof attached oligonucleotide sequences in a controlled manner. In someinstances, one or more oligonucleotide molecules (or other biomolecules)may be covalently tethered to an existing polymer layer after the layerhas been deposited on a surface.

FIG. 5 provides a schematic illustration of one non-limiting example ofcoupling a branched polymer comprising covalently-attachedoligonucleotide primers to a layered hydrophilic surface such as the oneillustrated in FIG. 3. In this example, a branched polymer comprisingtwo different oligonucleotide primers (represented by dotted and dashedlines) and amine-reactive NHS groups is coupled to the primary amines ofthe previous layer to create a multilayered, three-dimensionalhydrophilic surface comprising a controlled surface density of tetheredoligonucleotide primers.

The attachment chemistry used to graft a first chemically-modified layerto a support surface will generally be dependent on both the materialfrom which the support is fabricated and the chemical nature of thelayer. In some instances, the first layer may be covalently attached tothe support surface. In some instances, the first layer may benon-covalently attached, e.g., adsorbed to the surface throughnon-covalent interactions such as electrostatic interactions, hydrogenbonding, or van der Waals interactions between the surface and themolecular components of the first layer. In either case, the substratesurface may be treated prior to attachment or deposition of the firstlayer. Any of a variety of surface preparation techniques known to thoseof skill in the art may be used to clean or treat the support surface.For example, glass or silicon surfaces may be acid-washed using aPiranha solution (a mixture of sulfuric acid (H₂SO₄) and hydrogenperoxide (H₂O₂)) and/or cleaned using an oxygen plasma treatment method.

Silane chemistries constitute one non-limiting approach for covalentlymodifying the silanol groups on glass or silicon surfaces to attach morereactive functional groups (e.g., amines or carboxyl groups), which maythen be used in coupling linker molecules (e.g., linear hydrocarbonmolecules of various lengths, such as C6, C12, C18 hydrocarbons, orlinear polyethylene glycol (PEG) molecules) or layer molecules (e.g.,branched PEG molecules or other polymers) to the surface. Examples ofsuitable silanes that may be used in creating any of the disclosed lowbinding support surfaces include, but are not limited to,(3-Aminopropyl) trimethoxysilane (APTMS), (3-Aminopropyl)triethoxysilane (APTES), any of a variety of PEG-silanes (e.g.,comprising molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEGsilane (i.e., comprising a free amino functional group), maleimide-PEGsilane, biotin-PEG silane, and the like.

Any of a variety of molecules known to those of skill in the artincluding, but not limited to, amino acids, peptides, nucleotides,oligonucleotides, other monomers or polymers, or combinations thereofmay be used in creating the one or more chemically-modified layers onthe support surface, where the choice of components used may be variedto alter one or more properties of the support surface, e.g., thesurface density of functional groups and/or tethered oligonucleotideprimers, the hydrophilicity/hydrophobicity of the support surface, orthe three three-dimensional nature (i.e., “thickness”) of the supportsurface. Examples of preferred polymers that may be used to create oneor more layers of low non-specific binding material in any of thedisclosed support surfaces include, but are not limited to, polyethyleneglycol (PEG) of various molecular weights and branching structures,streptavidin, polyacrylamide, polyester, dextran, poly-lysine, andpoly-lysine copolymers, or any combination thereof. Examples ofconjugation chemistries that may be used to graft one or more layers ofmaterial (e.g. polymer layers) to the support surface and/or tocross-link the layers to each other include, but are not limited to,biotin-streptavidin interactions (or variations thereof), his tag—Ni/NTAconjugation chemistries, methoxy ether conjugation chemistries,carboxylate conjugation chemistries, amine conjugation chemistries, NHSesters, maleimides, thiol, epoxy, azide, hydrazide, alkyne, isocyanate,and silane.

One or more layers of a multi-layered surface may comprise a branchedpolymer or may be linear. Examples of suitable branched polymersinclude, but are not limited to, branched PEG, branched poly(vinylalcohol) (branched PVA), branched poly(vinyl pyridine), branchedpoly(vinyl pyrrolidone) (branched PVP), branched), poly(acrylic acid)(branched PAA), branched polyacrylamide, branchedpoly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methylmethacrylate) (branched PMA), branched poly(-hydroxylethyl methacrylate)(branched PHEMA), branched poly(oligo(ethylene glycol) methyl ethermethacrylate) (branched POEGMA), branched polyglutamic acid (branchedPGA), branched poly-lysine, branched poly-glucoside, and dextran.

In some instances, the branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein maycomprise at least 4 branches, at least 5 branches, at least 6 branches,at least 7 branches, at least 8 branches, at least 9 branches, at least10 branches, at least 12 branches, at least 14 branches, at least 16branches, at least 18 branches, at least 20 branches, at least 22branches, at least 24 branches, at least 26 branches, at least 28branches, at least 30 branches, at least 32 branches, at least 34branches, at least 36 branches, at least 38 branches, or at least 40branches. Molecules often exhibit a ‘power of 2’ number of branches,such as 2, 4, 8, 16, 32, 64, or 128 branches.

Exemplary PEG multilayers include PEG (8,16,8) (8 arm, 16 arm, 8 arm)?on PEG-amine-APTES. Similar concentrations were observed for 3-layermulti-arm PEG (8 arm, 16arm, 8arm) and (8arm, 64arm, 8arm) onPEG-amine-APTES exposed to 8 uM primer, and 3-layer multi-arm PEG (8arm,8arm, 8arm) using star-shape PEG-amine to replace 16 arm and 64 arm PEGmultilayers having comparable first, second and third PEG layers arealso contemplated.

Linear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may have amolecular weight of at least 500, at least 1,000, at least 1,500, atleast 2,000, at least 2,500, at least 3,000, at least 3,500, at least4,000, at least 4,500, at least 5,000, at least 7,500, at least 10,000,at least 12,500, at least 15,000, at least 17,500, at least 20,000, atleast 25,000, at least 30,000, at least 35,000, at least 40,000, atleast 45,000, or at least 50,000 Daltons. In some instances, the linear,branched, or multi-branched polymers used to create one or more layersof any of the multi-layered surfaces disclosed herein may have amolecular weight of at most 50,000, at most 45,000, at most 40,000, atmost 35,000, at most 30,000, at most 25,000, at most 20,000, at most17,500, at most 15,000, at most 12,500, at most 10,000, at most 7,500,at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, or atmost 500 Daltons. Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the molecular weight oflinear, branched, or multi-branched polymers used to create one or morelayers of any of the multi-layered surfaces disclosed herein may rangefrom about 1,500 to about 20,000 Daltons. Those of skill in the art willrecognize that the molecular weight of linear, branched, ormulti-branched polymers used to create one or more layers of any of themulti-layered surfaces disclosed herein may have any value within thisrange, e.g., about 1,260 Daltons.

In some instances, e.g., wherein at least one layer of a multi-layeredsurface comprises a branched polymer, the number of covalent bondsbetween a branched polymer molecule of the layer being deposited andmolecules of the previous layer may range from about one covalentlinkage per molecule and about 32 covalent linkages per molecule. Insome instances, the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the previous layer may be atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 12, at least 14,at least 16, at least 18, at least 20, at least 22, at least 24, atleast 26, at least 28, at least 30, or at least 32, or more than 32covalent linkages per molecule. In some instances, the number ofcovalent bonds between a branched polymer molecule of the new layer andmolecules of the previous layer may be at most 32, at most 30, at most28, at most 26, at most 24, at most 22, at most 20, at most 18, at most16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7,at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the number of covalent bonds between abranched polymer molecule of the new layer and molecules of the previouslayer may range from about 4 to about 16. Those of skill in the art willrecognize that the number of covalent bonds between a branched polymermolecule of the new layer and molecules of the previous layer may haveany value within this range, e.g., about 11 in some instances, or anaverage number of about 4.6 in other instances.

Any reactive functional groups that remain following the coupling of amaterial layer to the support surface may optionally be blocked bycoupling a small, inert molecule using a high yield coupling chemistry.For example, in the case that amine coupling chemistry is used to attacha new material layer to the previous one, any residual amine groups maysubsequently be acetylated or deactivated by coupling with a small aminoacid such as glycine.

The number of layers of low non-specific binding material, e.g., ahydrophilic polymer material, deposited on the surface of the disclosedlow binding supports may range from 1 to about 10. In some instances,the number of layers is at least 1, at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10. In some instances, the number of layers may be at most 10, at most9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, atmost 2, or at most 1. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the number of layersmay range from about 2 to about 4. In some instances, all of the layersmay comprise the same material. In some instances, each layer maycomprise a different material. In some instances, the plurality oflayers may comprise a plurality of materials. In some instances at leastone layer may comprise a branched polymer. In some instance, all of thelayers may comprise a branched polymer.

One or more layers of low non-specific binding material may in somecases be deposited on and/or conjugated to the substrate surface using apolar protic solvent, a polar aprotic solvent, a nonpolar solvent, orany combination thereof. In some instances the solvent used for layerdeposition and/or coupling may comprise an alcohol (e.g., methanol,ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile,dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), etc.), water, anaqueous buffer solution (e.g., phosphate buffer, phosphate bufferedsaline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or anycombination thereof. In some instances, an organic component of thesolvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% of the total, or any percentage spanned or adjacent to the rangeherein, with the balance made up of water or an aqueous buffer solution.In some instances, an aqueous component of the solvent mixture used maycomprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or anypercentage spanned or adjacent to the range herein, with the balancemade up of an organic solvent. The pH of the solvent mixture used may beless than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greaterthan 10, or any value spanned or adjacent to the range described herein.

In some instances, one or more layers of low non-specific bindingmaterial may be deposited on and/or conjugated to the substrate surfaceusing a mixture of organic solvents, wherein the dielectric constant ofat least once component is less than 40 and constitutes at least 50% ofthe total mixture by volume. In some instances, the dielectric constantof the at least one component may be less than 10, less than 20, lessthan 30, less than 40. In some instances, the at least one componentconstitutes at least 20%, at least 30%, at least 40%, at least 50%, atleast 50%, at least 60%, at least 70%, or at least 80% of the totalmixture by volume.

As noted, the low non-specific binding supports of the presentdisclosure exhibit reduced non-specific binding of proteins, nucleicacids, and other components of the hybridization and/or amplificationformulation used for solid-phase nucleic acid amplification. The degreeof non-specific binding exhibited by a given support surface may beassessed either qualitatively or quantitatively. For example, in someinstances, exposure of the surface to fluorescent dyes (e.g., Cy3, Cy5,etc.), fluorescently-labeled nucleotides, fluorescently-labeledoligonucleotides, and/or fluorescently-labeled proteins (e.g.polymerases) under a standardized set of conditions, followed by aspecified rinse protocol and fluorescence imaging may be used as aqualitative tool for comparison of non-specific binding on supportscomprising different surface formulations. In some instances, exposureof the surface to fluorescent dyes, fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, and/or fluorescently-labeledproteins (e.g. polymerases) under a standardized set of conditions,followed by a specified rinse protocol and fluorescence imaging may beused as a quantitative tool for comparison of non-specific binding onsupports comprising different surface formulations—provided that carehas been taken to ensure that the fluorescence imaging is performedunder conditions where fluorescence signal is linearly related (orrelated in a predictable manner) to the number of fluorophores on thesupport surface (e.g., under conditions where signal saturation and/orself-quenching of the fluorophore is not an issue) and suitablecalibration standards are used. In some instances, other techniquesknown to those of skill in the art, for example, radioisotope labelingand counting methods may be used for quantitative assessment of thedegree to which non-specific binding is exhibited by the differentsupport surface formulations of the present disclosure.

Some surfaces disclosed herein exhibit a ratio of specific tononspecific binding of a fluorophore such as Cy3 of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,50, 75, 100, or greater than 100, or any intermediate value spanned bythe range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to nonspecific fluorescence of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein.

As noted, in some instances, the degree of non-specific bindingexhibited by the disclosed low-binding supports may be assessed using astandardized protocol for contacting the surface with a labeled protein(e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, areverse transcriptase, a helicase, a single-stranded binding protein(SSB), etc., or any combination thereof), a labeled nucleotide, alabeled oligonucleotide, etc., under a standardized set of incubationand rinse conditions, followed be detection of the amount of labelremaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label known to one of skill in the art. In someinstances, the degree of non-specific binding exhibited by a givensupport surface formulation may thus be assessed in terms of the numberof non-specifically bound protein molecules (or other molecules) perunit area. In some instances, the low-binding supports of the presentdisclosure may exhibit non-specific protein binding (or non-specificbinding of other specified molecules, e.g., Cy3 dye) of less than 0.001molecule per μm², less than 0.01 molecule per μm², less than 0.1molecule per μm², less than 0.25 molecule per μm², less than 0.5molecule per μm², less than 1 molecule per μm², less than 10 moleculesper μm², less than 100 molecules per μm², or less than 1,000 moleculesper μm². Those of skill in the art will realize that a given supportsurface of the present disclosure may exhibit non-specific bindingfalling anywhere within this range, for example, of less than 86molecules per μm². For example, some modified surfaces disclosed hereinexhibit nonspecific protein binding of less than 0.5 molecule/μm²following contact with a 1 uM solution of Cy3 labeled streptavidin (GEAmersham) in phosphate buffered saline (PBS) buffer for 15 minutes,followed by 3 rinses with deionized water. Some modified surfacesdisclosed herein exhibit nonspecific binding of Cy3 dye molecules ofless than 0.25 molecules per um². In independent nonspecific bindingassays, 1 uM labeled Cy3 SA (ThermoFisher), 1 uM Cy5 SA dye(ThermoFisher), 10 uM Aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10uM Aminoallyl-dUTP-ATTO-Rho 11 (Jena Biosciences), 10 uMAminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 uM7-Propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences, and 10 uM7-Propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated onthe low binding substrates at 37° C. for 15 minutes in a 384 well plateformat. Each well was rinsed 2-3× with 50 ul deionized RNase/DNase Freewater and 2-3× with 25 mM ACES buffer pH 7.4. The 384 well plates wereimaged on a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, Pa.)instrument using the Cy3, AF555, or Cy5 filter sets (according to dyetest performed) as specified by the manufacturer at a PMT gain settingof 800 and resolution of 50-100 μm. For higher resolution imaging,images were collected on an Olympus IX83 microscope (Olympus Corp.,Center Valley, Pa.) with a total internal reflectance fluorescence(TIRF) objective (20×, 0.75 NA or 100λ, 1.5 NA, Olympus), an sCMOS Andorcamera (Zyla 4.2), and excitation wavelengths of 532 nm or 635 nm.Dichroic mirrors were purchased from Semrock (IDEX Health & Science,LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633 nm dichroicreflectors/beamsplitters, and band pass filters were chosen as 532 LP or645 LP concordant with the appropriate excitation wavelength. Somemodified surfaces disclosed herein exhibit nonspecific binding of dyemolecules of less than 0.25 molecules per μm².

In some instances, the surfaces disclosed herein exhibit a ratio ofspecific to nonspecific binding of a fluorophore such as Cy3 of at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate valuespanned by the range herein. In some instances, the surfaces disclosedherein exhibit a ratio of specific to nonspecific fluorescence signalsfor a fluorophore such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, orgreater than 100, or any intermediate value spanned by the range herein.

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or morethan 50 specific dye molecules attached per molecule nonspecificallyadsorbed. Similarly, when subjected to an excitation energy,low-background surfaces consistent with the disclosure herein to whichfluorophores, e.g., Cy3, have been attached may exhibit ratios ofspecific fluorescence signal (e.g., arising from Cy3-labeledoligonucleotides attached to the surface) to non-specific adsorbed dyefluorescence signals of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces may be assessed,for example, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome instances, a static contact angle may be determined. In someinstances, an advancing or receding contact angle may be determined. Insome instances, the water contact angle for the hydrophilic, low-bindingsupport surfaced disclosed herein may range from about 0 degrees toabout 50 degrees. In some instances, the water contact angle for thehydrophilic, low-binding support surfaced disclosed herein may no morethan 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. Inmany cases the contact angle is no more than any value within thisrange, e.g., no more than 40 degrees. Those of skill in the art willrealize that a given hydrophilic, low-binding support surface of thepresent disclosure may exhibit a water contact angle having a value ofanywhere within this range, e.g., about 27 degrees.

In some instances, the hydrophilic surfaces disclosed herein facilitatereduced wash times for bioassays, often due to reduced nonspecificbinding of biomolecules to the low-binding surfaces. In some instances,adequate wash steps may be performed in less than 60, 50, 40, 30, 20,15, 10, or less than 10 seconds. For example, in some instances adequatewash steps may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantimprovement in stability or durability to prolonged exposure to solventsand elevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. For example, in some instances, the stability ofthe disclosed surfaces may be tested by fluorescently labeling afunctional group on the surface, or a tethered biomolecule (e.g., anoligonucleotide primer) on the surface, and monitoring fluorescencesignal before, during, and after prolonged exposure to solvents andelevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. In some instances, the degree of change in thefluorescence used to assess the quality of the surface may be less than1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a time period of 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50hours, or 100 hours of exposure to solvents and/or elevated temperatures(or any combination of these percentages as measured over these timeperiods). In some instances, the degree of change in the fluorescenceused to assess the quality of the surface may be less than 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeatedexposure to solvent changes and/or changes in temperature (or anycombination of these percentages as measured over this range of cycles).

In some instances, the surfaces disclosed herein may exhibit a highratio of specific signal to nonspecific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greater than asignal of an adjacent unpopulated region of the surface. Similarly, somesurfaces exhibit an amplification signal that is at least 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greaterthan a signal of an adjacent amplified nucleic acid population region ofthe surface.

Fluorescence excitation energies vary among particular fluorophores andprotocols, and may range in excitation wavelength from less than 400 nmto over 800 nm, consistent with fluorophore selection or otherparameters of use of a surface disclosed herein.

Accordingly, low background surfaces as disclosed herein exhibit lowbackground fluorescence signals or high contrast to noise (CNR) ratiosrelative to known surfaces in the art. For example, in some instances,the background fluorescence of the surface at a location that isspatially distinct or removed from a labeled feature on the surface(e.g., a labeled spot, cluster, discrete region, sub-section, or subsetof the surface) comprising a hybridized cluster of nucleic acidmolecules, or a clonally-amplified cluster of nucleic acid moleculesproduced by, e.g., 20 cycles of nucleic acid amplification viathermocycling, may be no more than 20×, 10×, 5×, 2×, 1×, 0.5×, 0.1×, orless than 0.1× greater than the background fluorescence measured at thatsame location prior to performing said hybridization or said 20 cyclesof nucleic acid amplification.

In some instances, fluorescence images of the disclosed low backgroundsurfaces when used in nucleic acid hybridization or amplificationapplications to create clusters of hybridized or clonally-amplifiednucleic acid molecules (e.g., that have been directly or indirectlylabeled with a fluorophore) exhibit contrast-to-noise ratios (CNRs) ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than250.

Oligonucleotide primers and adapter sequences: In general, at least onelayer of the one or more layers of low non-specific binding material maycomprise functional groups for covalently or non-covalently attachingoligonucleotide molecules, e.g., adapter or primer sequences, or the atleast one layer may already comprise covalently or non-covalentlyattached oligonucleotide adapter or primer sequences at the time that itis deposited on the support surface. In some instances, theoligonucleotides tethered to the polymer molecules of at least one thirdlayer may be distributed at a plurality of depths throughout the layer.

In some instances, the oligonucleotide adapter or primer molecules arecovalently coupled to the polymer in solution, i.e., prior to couplingor depositing the polymer on the surface. In some instances, theoligonucleotide adapter or primer molecules are covalently coupled tothe polymer after it has been coupled to or deposited on the surface. Insome instances, at least one hydrophilic polymer layer comprises aplurality of covalently-attached oligonucleotide adapter or primermolecules. In some instances, at least two, at least three, at leastfour, or at least five layers of hydrophilic polymer comprise aplurality of covalently-attached adapter or primer molecules.

In some instances, the oligonucleotide adapter or primer molecules maybe coupled to the one or more layers of hydrophilic polymer using any ofa variety of suitable conjugation chemistries known to those of skill inthe art. For example, the oligonucleotide adapter or primer sequencesmay comprise moieties that are reactive with amine groups, carboxylgroups, thiol groups, and the like. Examples of suitable amine-reactiveconjugation chemistries that may be used include, but are not limitedto, reactions involving isothiocyanate, isocyanate, acyl azide, NHSester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane,carbonate, aryl halide, imidoester, carbodiimide, anhydride, andfluorophenyl ester groups. Examples of suitable carboxyl-reactiveconjugation chemistries include, but are not limited to, reactionsinvolving carbodiimide compounds, e.g., water soluble EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide⋅HCL). Examples ofsuitable sulfydryl-reactive conjugation chemistries include maleimides,haloacetyls and pyridyl disulfides.

One or more types of oligonucleotide molecules may be attached ortethered to the support surface. In some instances, the one or moretypes of oligonucleotide adapters or primers may comprise spacersequences, adapter sequences for hybridization to adapter-ligatedtemplate library nucleic acid sequences, forward amplification primers,reverse amplification primers, sequencing primers, and/or molecularbarcoding sequences, or any combination thereof. In some instances, 1primer or adapter sequence may be tethered to at least one layer of thesurface. In some instances, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 different primer or adapter sequences may be tethered to atleast one layer of the surface.

In some instances, the tethered oligonucleotide adapter and/or primersequences may range in length from about 10 nucleotides to about 100nucleotides. In some instances, the tethered oligonucleotide adapterand/or primer sequences may be at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, or at least 100 nucleotides in length. In some instances, thetethered oligonucleotide adapter and/or primer sequences may be at most100, at most 90, at most 80, at most 70, at most 60, at most 50, at most40, at most 30, at most 20, or at most 10 nucleotides in length. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the length of the tethered oligonucleotide adapter and/orprimer sequences may range from about 20 nucleotides to about 80nucleotides. Those of skill in the art will recognize that the length ofthe tethered oligonucleotide adapter and/or primer sequences may haveany value within this range, e.g., about 24 nucleotides.

In some instances, the tethered adapter or primer sequences may comprisemodifications designed to facilitate the specificity and efficiency ofnucleic acid amplification as performed on the low-binding supports. Forexample, in some instances the primer may comprise polymerase stoppoints such that the stretch of primer sequence between the surfaceconjugation point and the modification site is always in single-strandedform and functions as a loading site for 5′ to 3′ helicases in somehelicase-dependent isothermal amplification methods. Other examples ofprimer modifications that may be used to create polymerase stop pointsinclude, but are not limited to, an insertion of a PEG chain into thebackbone of the primer between two nucleotides towards the 5′ end,insertion of an abasic nucleotide (i.e., a nucleotide that has neither apurine nor a pyrimidine base), or a lesion site which can be bypassed bythe helicase.

As will be discussed further in the examples below, it may be desirableto vary the surface density of tethered oligonucleotide adapters orprimers on the support surface and/or the spacing of the tetheredadapters or primers away from the support surface (e.g., by varying thelength of a linker molecule used to tether the adaptors or primers tothe surface) in order to “tune” the support for optimal performance whenusing a given amplification method. As noted below, adjusting thesurface density of tethered oligonucleotide adapters or primers mayimpact the level of specific and/or non-specific amplification observedon the support in a manner that varies according to the amplificationmethod selected. In some instances, the surface density of tetheredoligonucleotide adapters or primers may be varied by adjusting the ratioof molecular components used to create the support surface. For example,in the case that an oligonucleotide primer—PEG conjugate is used tocreate the final layer of a low-binding support, the ratio of theoligonucleotide primer—PEG conjugate to a non-conjugated PEG moleculemay be varied. The resulting surface density of tethered primermolecules may then be estimated or measured using any of a variety oftechniques known to those of skill in the art. Examples include, but arenot limited to, the use of radioisotope labeling and counting methods,covalent coupling of a cleavable molecule that comprises anoptically-detectable tag (e.g., a fluorescent tag) that may be cleavedfrom a support surface of defined area, collected in a fixed volume ofan appropriate solvent, and then quantified by comparison offluorescence signals to that for a calibration solution of known opticaltag concentration, or using fluorescence imaging techniques providedthat care has been taken with the labeling reaction conditions and imageacquisition settings to ensure that the fluorescence signals arelinearly related to the number of fluorophores on the surface (e.g.,that there is no significant self-quenching of the fluorophores on thesurface).

In some instances, the resultant surface density of oligonucleotideadapters or primers on the low binding support surfaces of the presentdisclosure may range from about 100 primer molecules per μm² to about1,000,000 primer molecules per μm². In some instances, the surfacedensity of oligonucleotide adapters or primers may be at least 100, atleast 200, at least 300, at least 400, at least 500, at least 600, atleast 700, at least 800, at least 900, at least 1,000, at least 1,500,at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000,at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000,at least 20,000, at least 25,000, at least 30,000, at least 35,000, atleast 40,000, at least 45,000, at least 50,000, at least 55,000, atleast 60,000, at least 65,000, at least 70,000, at least 75,000, atleast 80,000, at least 85,000, at least 90,000, at least 95,000, atleast 100,000, at least 150,000, at least 200,000, at least 250,000, atleast 300,000, at least 350,000, at least 400,000, at least 450,000, atleast 500,000, at least 550,000, at least 600,000, at least 650,000, atleast 700,000, at least 750,000, at least 800,000, at least 850,000, atleast 900,000, at least 950,000, or at least 1,000,000 molecules perμm². In some instances, the surface density of oligonucleotide adaptersor primers may be at most 1,000,000, at most 950,000, at most 900,000,at most 850,000, at most 800,000, at most 750,000, at most 700,000, atmost 650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, atmost 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000,at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most800, at most 700, at most 600, at most 500, at most 400, at most 300, atmost 200, or at most 100 molecules per μm². Any of the lower and uppervalues described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe surface density of adapters or primers may range from about 10,000molecules per μm² to about 100,000 molecules per μm². Those of skill inthe art will recognize that the surface density of adapter or primermolecules may have any value within this range, e.g., about 3,800molecules per μm² in some instances, or about 455,000 molecules per μm²in other instances. In some instances, as will be discussed furtherbelow, the surface density of template library nucleic acid sequences(e.g., sample DNA molecules) initially hybridized to adapter or primersequences on the support surface may be less than or equal to thatindicated for the surface density of tethered oligonucleotide primers.In some instances, as will also be discussed further below, the surfacedensity of clonally-amplified template library nucleic acid sequenceshybridized to adapter or primer sequences on the support surface mayspan the same range or a different range as that indicated for thesurface density of tethered oligonucleotide adapters or primers.

Local surface densities of adapter or primer molecules as listed abovedo not preclude variation in density across a surface, such that asurface may comprise a region having an oligo density of, for example,500,000/um², while also comprising at least a second region having asubstantially different local density.

Hybridization of nucleic acid molecules to low-binding supports: In someaspects of the present disclosure, hybridization buffer formulations aredescribed which, in combination with the disclosed low-binding supports,provide for improved hybridization rates, hybridization specificity (orstringency), and hybridization efficiency (or yield). As used herein,hybridization specificity is a measure of the ability of tetheredadapter sequences, primer sequences, or oligonucleotide sequences ingeneral to correctly hybridize only to completely complementarysequences, while hybridization efficiency is a measure of the percentageof total available tethered adapter sequences, primer sequences, oroligonucleotide sequences in general that are hybridized tocomplementary sequences.

Improved hybridization specificity and/or efficiency may be achievedthrough optimization of the hybridization buffer formulation used withthe disclosed low-binding surfaces, and will be discussed in more detailin the examples below. Examples of hybridization buffer components thatmay be adjusted to achieve improved performance include, but are notlimited to, buffer type, organic solvent mixtures, buffer pH, bufferviscosity, detergents and zwitterionic components, ionic strength(including adjustment of both monovalent and divalent ionconcentrations), antioxidants and reducing agents, carbohydrates, BSA,polyethylene glycol, dextran sulfate, betaine, other additives, and thelike.

By way of non-limiting example, suitable buffers for use in formulatinga hybridization buffer may include, but are not limited to, phosphatebuffered saline (PBS), succinate, citrate, histidine, acetate, Tris,TAPS, MOPS, PIPES, HEPES, MES, and the like. The choice of appropriatebuffer will generally be dependent on the target pH of the hybridizationbuffer solution. In general, the desired pH of the buffer solution willrange from about pH 4 to about pH 8.4. In some embodiments, the bufferpH may be at least 4.0, at least 4.5, at least 5.0, at least 5.5, atleast 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, atleast 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, atleast 8.0, at least 8.2, or at least 8.4. In some embodiments, thebuffer pH may be at most 8.4, at most 8.2, at most 8.0, at most 7.8, atmost 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at most6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at most 5.0, atmost 4.5, or at most 4.0. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances, the desired pH mayrange from about 6.4 to about 7.2. Those of skill in the art willrecognize that the buffer pH may have any value within this range, forexample, about 7.25.

Suitable detergents for use in hybridization buffer formulation include,but are not limited to, zitterionic detergents (e.g.,1-Dodecanoyl-sn-glycero-3-phosphocholine,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,NDimethylmyristylammonio) propanesulfonate, ASB-C80, C7BzO, CHAPS,CHAPS hydrate, CHAPSO, DDMAB, Dimethylethylammoniumpropane sulfonate,N,N-Dimethyldodecylamine Noxide,N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, orN-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and anionic,cationic, and non-ionic detergents. Examples of nonionic detergentsinclude poly(oxyethylene) ethers and related polymers (e.g. Brij®,TWEEN®, TRITON®, TRITON X-100 and IGEPAL® CA-630), bile salts, andglycosidic detergents.

The use of the disclosed low-binding supports either alone or incombination with optimized buffer formulations may yield relativehybridization rates that range from about 2× to about 20× faster thanthat for a conventional hybridization protocol. In some instances, therelative hybridization rate may be at least 2×, at least 3×, at least4×, at least 5×, at least 6×, at least 7×, at least 8×, at least 9×, atleast 10×, at least 12×, at least 14×, at least 16×, at least 18×, atleast 20×, at least 25×, at least 30×, or at least 40× that for aconventional hybridization protocol.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized buffer formulations may yield totalhybridization reaction times (i.e., the time required to reach 90%, 95%,98%, or 99% completion of the hybridization reaction) of less than 60minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10minutes, or 5 minutes for any of these completion metrics.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized buffer formulations may yield improvedhybridization specificity compared to that for a conventionalhybridization protocol. In some instances, the hybridization specificitythat may be achieved is better than 1 base mismatch in 10 hybridizationevents, 1 base mismatch in 20 hybridization events, 1 base mismatch in30 hybridization events, 1 base mismatch in 40 hybridization events, 1base mismatch in 50 hybridization events, 1 base mismatch in 75hybridization events, 1 base mismatch in 100 hybridization events, 1base mismatch in 200 hybridization events, 1 base mismatch in 300hybridization events, 1 base mismatch in 400 hybridization events, 1base mismatch in 500 hybridization events, 1 base mismatch in 600hybridization events, 1 base mismatch in 700 hybridization events, 1base mismatch in 800 hybridization events, 1 base mismatch in 900hybridization events, 1 base mismatch in 1,000 hybridization events, 1base mismatch in 2,000 hybridization events, 1 base mismatch in 3,000hybridization events, 1 base mismatch in 4,000 hybridization events, 1base mismatch in 5,000 hybridization events, 1 base mismatch in 6,000hybridization events, 1 base mismatch in 7,000 hybridization events, 1base mismatch in 8,000 hybridization events, 1 base mismatch in 9,000hybridization events, or 1 base mismatch in 10,000 hybridization events.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized buffer formulations may yield improvedhybridization efficiency (e.g., the fraction of availableoligonucleotide primers on the support surface that are successfullyhybridized with target oligonucleotide sequences) compared to that for aconventional hybridization protocol. In some instances, thehybridization efficiency that may be achieved is better than 50%, 60%,70%, 80%, 85%, 90%, 95%, 98%, or 99% for any of the input targetoligonucleotide concentrations specified below and in any of thehybridization reaction times specified above. In some instances, e.g.,wherein the hybridization efficiency is less than 100%, the resultingsurface density of target nucleic acid sequences hybridized to thesupport surface may be less than the surface density of oligonucleotideadapter or primer sequences on the surface.

In some instances, use of the disclosed low-binding supports for nucleicacid hybridization (or amplification) applications using conventionalhybridization (or amplification) protocols, or optimized hybridization(or amplification) protocols may lead to a reduced requirement for theinput concentration of target (or sample) nucleic acid moleculescontacted with the support surface. For example, in some instances, thetarget (or sample) nucleic acid molecules may be contacted with thesupport surface at a concentration ranging from about 10 pM to about 1μM (i.e., prior to annealing or amplification). In some instances, thetarget (or sample) nucleic acid molecules may be administered at aconcentration of at least 10 pM, at least 20 pM, at least 30 pM, atleast 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700pM, at least 800 pM, at least 900 pM, at least 1 nM, at least 10 nM, atleast 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, atleast 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, atleast 600 nM, at leasy 700 nM, at least 800 nM, at least 900 nM, or atleast 1 μM. In some instances, the target (or sample) nucleic acidmolecules may be administered at a concentration of at most 1 μM, atmost 900 nM, at most 800 nm, at most 700 nM, at most 600 nM, at most 500nM, at most 400 nM, at most 300 nM, at most 200 nM, at most 100 nM, atmost 90 nM, at most 80 nM, at most 70 nM, at most 60 nM, at most 50 nM,at most 40 nM, at most 30 nM, at most 20 nM, at most 10 nM, at most 1nM, at most 900 pM, at most 800 pM, at most 700 pM, at most 600 pM, atmost 500 pM, at most 400 pM, at most 300 pM, at most 200 pM, at most 100pM, at most 90 pM, at most 80 pM, at most 70 pM, at most 60 pM, at most50 pM, at most 40 pM, at most 30 pM, at most 20 pM, or at most 10 pM.Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the target (or sample) nucleic acid moleculesmay be administered at a concentration ranging from about 90 pM to about200 nM. Those of skill in the art will recognize that the target (orsample) nucleic acid molecules may be administered at a concentrationhaving any value within this range, e.g., about 855 nM.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized hybridization buffer formulations mayresult in a surface density of hybridized target (or sample)oligonucleotide molecules (i.e., prior to performing any subsequentsolid-phase or clonal amplification reaction) ranging from about fromabout 0.0001 target oligonucleotide molecules per μm² to about 1,000,000target oligonucleotide molecules per μm². In some instances, the surfacedensity of hybridized target oligonucleotide molecules may be at least0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01,at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 200,at least 300, at least 400, at least 500, at least 600, at least 700, atleast 800, at least 900, at least 1,000, at least 1,500, at least 2,000,at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500,at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least9,000, at least 9,500, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, or at least 1,000,000 molecules per μm². Insome instances, the surface density of hybridized target oligonucleotidemolecules may be at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, atmost 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000,at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most800, at most 700, at most 600, at most 500, at most 400, at most 300, atmost 200, at most 100, at most 90, at most 80, at most 70, at most 60,at most 50, at most 40, at most 30, at most 20, at most 10, at most 5,at most 1, at most 0.5, at most 0.1, at most 0.05, at most 0.01, at most0.005, at most 0.001, at most 0.0005, or at most 0.0001 molecules perμm². Any of the lower and upper values described in this paragraph maybe combined to form a range included within the present disclosure, forexample, in some instances the surface density of hybridized targetoligonucleotide molecules may range from about 3,000 molecules per μm²to about 20,000 molecules per μm². Those of skill in the art willrecognize that the surface density of hybridized target oligonucleotidemolecules may have any value within this range, e.g., about 2,700molecules per μm².

Stated differently, in some instances the use of the disclosedlow-binding supports alone or in combination with optimizedhybridization buffer formulations may result in a surface density ofhybridized target (or sample) oligonucleotide molecules (i.e., prior toperforming any subsequent solid-phase or clonal amplification reaction)ranging from about 100 hybridized target oligonucleotide molecules permm² to about 1×10⁷ oligonucleotide molecules per mm² or from about 100hybridized target oligonucleotide molecules per mm² to about 1×10¹²hybridized target oligonucleotide molecules per mm². In some instances,the surface density of hybridized target oligonucleotide molecules maybe at least 100, at least 500, at least 1,000, at least 4,000, at least5,000, at least 6,000, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, at least100,000, at least 150,000, at least 200,000, at least 250,000, at least300,000, at least 350,000, at least 400,000, at least 450,000, at least500,000, at least 550,000, at least 600,000, at least 650,000, at least700,000, at least 750,000, at least 800,000, at least 850,000, at least900,000, at least 950,000, at least 1,000,000, at least 5,000,000, atleast 1×10⁷, at least 5×10⁷, at least 1×10⁸, at least 5×10⁸, at least1×10⁹, at least 5×10⁹, at least 1×10¹⁰, at least 5×10¹⁰, at least1×10¹¹, at least 5×10¹¹, or at least 1×10¹² molecules per mm². In someinstances, the surface density of hybridized target oligonucleotidemolecules may be at most 1×10¹², at most 5×10¹¹, at most 1×10¹¹, at most5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most 1×10⁹, at most 5×10⁸, atmost 1×10⁸, at most 5×10⁷, at most 1×10⁷, at most 5,000,000, at most1,000,000, at most 950,000, at most 900,000, at most 850,000, at most800,000, at most 750,000, at most 700,000, at most 650,000, at most600,000, at most 550,000, at most 500,000, at most 450,000, at most400,000, at most 350,000, at most 300,000, at most 250,000, at most200,000, at most 150,000, at most 100,000, at most 95,000, at most90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000,at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000,at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most1,000, at most 500, or at most 100 molecules per mm². Any of the lowerand upper values described in this paragraph may be combined to form arange included within the present disclosure, for example, in someinstances the surface density of hybridized target oligonucleotidemolecules may range from about 5,000 molecules per mm² to about 50,000molecules per mm². Those of skill in the art will recognize that thesurface density of hybridized target oligonucleotide molecules may haveany value within this range, e.g., about 50,700 molecules per mm².

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) hybridized to the oligonucleotide adapter orprimer molecules attached to the low-binding support surface may rangein length from about 0.02 kilobases (kb) to about 20 kb or from about0.1 kilobases (kb) to about 20 kb. In some instances, the targetoligonucleotide molecules may be at least 0.001 kb, at least 0.005 kb,at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb inlength, at least 0.2 kb in length, at least 0.3 kb in length, at least0.4 kb in length, at least 0.5 kb in length, at least 0.6 kb in length,at least 0.7 kb in length, at least 0.8 kb in length, at least 0.9 kb inlength, at least 1 kb in length, at least 2 kb in length, at least 3 kbin length, at least 4 kb in length, at least 5 kb in length, at least 6kb in length, at least 7 kb in length, at least 8 kb in length, at least9 kb in length, at least 10 kb in length, at least 15 kb in length, atleast 20 kb in length, at least 30 kb in length, or at least 40 kb inlength, or any intermediate value spanned by the range described herein,e.g., at least 0.85 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-stranded,multimeric nucleic acid molecules further comprising repeats of aregularly occurring monomer unit. In some instances, the single-strandedor double-stranded, multimeric nucleic acid molecules may be at least0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, atleast 0.05 kb, at least 0.1 kb in length, at least 0.2 kb in length, atleast 0.3 kb in length, at least 0.4 kb in length, at least 0.5 kb inlength, at least 1 kb in length, at least 2 kb in length, at least 3 kbin length, at least 4 kb in length, at least 5 kb in length, at least 6kb in length, at least 7 kb in length, at least 8 kb in length, at least9 kb in length, at least 10 kb in length, at least 15 kb in length, orat least 20 kb in length, at least 30 kb in length, or at least 40 kb inlength, or any intermediate value spanned by the range described herein,e.g., about 2.45 kb in length.

In some instances, the target (or sample) oligonucleotide molecules (ornucleic acid molecules) may comprise single-stranded or double-strandedmultimeric nucleic acid molecules comprising from about 2 to about 100copies of a regularly repeating monomer unit. In some instances, thenumber of copies of the regularly repeating monomer unit may be at least2, at least 3, at least 4, at least 5, at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 55, at least 60, at least 65, at least 70, atleast 75, at least 80, at least 85, at least 90, at least 95, and atleast 100. In some instances, the number of copies of the regularlyrepeating monomer unit may be at most 100, at most 95, at most 90, atmost 85, at most 80, at most 75, at most 70, at most 65, at most 60, atmost 55, at most 50, at most 45, at most 40, at most 35, at most 30, atmost 25, at most 20, at most 15, at most 10, at most 5, at most 4, atmost 3, or at most 2. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the number of copiesof the regularly repeating monomer unit may range from about 4 to about60. Those of skill in the art will recognize that the number of copiesof the regularly repeating monomer unit may have any value within thisrange, e.g., about 17. Thus, in some instances, the surface density ofhybridized target sequences in terms of the number of copies of a targetsequence per unit area of the support surface may exceed the surfacedensity of oligonucleotide primers even if the hybridization efficiencyis less than 100%.

Nucleic acid surface amplification (NASA): As used herein, the phrase“nucleic acid surface amplification” (NASA) is used interchangeably withthe phrase “solid-phase nucleic acid amplification” (or simply“solid-phase amplification”). In some aspects of the present disclosure,nucleic acid amplification formulations are described which, incombination with the disclosed low-binding supports, provide forimproved amplification rates, amplification specificity, andamplification efficiency. As used herein, specific amplification refersto amplification of template library oligonucleotide strands that havebeen tethered to the solid support either covalently or non-covalently.As used herein, non-specific amplification refers to amplification ofprimer-dimers or other non-template nucleic acids. As used herein,amplification efficiency is a measure of the percentage of tetheredoligonucleotides on the support surface that are successfully amplifiedduring a given amplification cycle or amplification reaction. Nucleicacid amplification performed on surfaces disclosed herein may obtainamplification efficiencies of at least 50%, 60%, 70%, 80%, 90%, 95%, orgreater than 95%, such as 98% or 99%.

Any of a variety of thermal cycling or isothermal nucleic acidamplification schemes may be used with the disclosed low-bindingsupports. Examples of nucleic acid amplification methods that may beutilized with the disclosed low-binding supports include, but are notlimited to, polymerase chain reaction (PCR), multiple displacementamplification (MDA), transcription-mediated amplification (TMA), nucleicacid sequence-based amplification (NASBA), strand displacementamplification (SDA), real-time SDA, bridge amplification, isothermalbridge amplification, rolling circle amplification, circle-to-circleamplification, helicase-dependent amplification, recombinase-dependentamplification, or single-stranded binding (SSB) protein-dependentamplification.

Often, improvements in amplification rate, amplification specificity,and amplification efficiency may be achieved using the disclosedlow-binding supports alone or in combination with formulations of theamplification reaction components. In addition to inclusion ofnucleotides, one or more polymerases, helicases, single-stranded bindingproteins, etc. (or any combination thereof), the amplification reactionmixture may be adjusted in a variety of ways to achieve improvedperformance including, but are not limited to, choice of buffer type,buffer pH, organic solvent mixtures, buffer viscosity, detergents andzwitterionic components, ionic strength (including adjustment of bothmonovalent and divalent ion concentrations), antioxidants and reducingagents, carbohydrates, BSA, polyethylene glycol, dextran sulfate,betaine, other additives, and the like.

The use of the disclosed low-binding supports alone or in combinationwith optimized amplification reaction formulations may yield increasedamplification rates compared to those obtained using conventionalsupports and amplification protocols. In some instances, the relativeamplification rates that may be achieved may be at least 2×, at least3×, at least 4×, at least 5×, at least 6×, at least 7×, at least 8×, atleast 9×, at least 10×, at least 12×, at least 14×, at least 16×, atleast 18×, or at least 20× that for use of conventional supports andamplification protocols for any of the amplification methods describedabove.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized buffer formulations may yield totalamplification reaction times (i.e., the time required to reach 90%, 95%,98%, or 99% completion of the amplification reaction) of less than 180mins, 120 mins, 90 min, 60 minutes, 50 minutes, 40 minutes, 30 minutes,20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, 50s, 40 s, 30 s, 20 s, or 1 Os for any of these completion metrics.

Some low-binding support surfaces disclosed herein exhibit a ratio ofspecific binding to nonspecific binding of a fluorophore such as Cy3 ofat least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1,75:1, 100:1, or greater than 100:1, or any intermediate value spanned bythe range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to nonspecific fluorescence signal for a fluorophore such asCy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1,40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediatevalue spanned by the range herein.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification buffer formulations mayenable faster amplification reaction times (i.e., the times required toreach 90%, 95%, 98%, or 99% completion of the amplification reaction) ofno more than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes,or 10 minutes. Similarly, use of the disclosed low-binding supportsalone or in combination with optimized buffer formulations may enableamplification reactions to be completed in some cases in no more than 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or no more than 30 cycles.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification reaction formulations mayyield increased specific amplification and/or decreased non-specificamplification compared to that obtained using conventional supports andamplification protocols. In some instances, the resulting ratio ofspecific amplification-to-non-specific amplification that may beachieved is at least 4:1 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1,40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1,600:1, 700:1, 800:1, 900:1, or 1,000:1.

In some instances, the use of the low-binding supports alone or incombination with optimized amplification reaction formulations may yieldincreased amplification efficiency compared to that obtained usingconventional supports and amplification protocols. In some instances,the amplification efficiency that may be achieved is better than 50%,60%, 70% 80%, 85%, 90%, 95%, 98%, or 99% in any of the amplificationreaction times specified above.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) hybridized to theoligonucleotide adapter or primer molecules attached to the low-bindingsupport surface may range in length from about 0.02 kilobases (kb) toabout 20 kb or from about 0.1 kilobases (kb) to about 20 kb. In someinstances, the clonally-amplified target oligonucleotide molecules maybe at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02kb, at least 0.05 kb, at least 0.1 kb in length, at least 0.2 kb inlength, at least 0.3 kb in length, at least 0.4 kb in length, at least0.5 kb in length, at least 1 kb in length, at least 2 kb in length, atleast 3 kb in length, at least 4 kb in length, at least 5 kb in length,at least 6 kb in length, at least 7 kb in length, at least 8 kb inlength, at least 9 kb in length, at least 10 kb in length, at least 15kb in length, or at least 20 kb in length, or any intermediate valuespanned by the range described herein, e.g., at least 0.85 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded, multimeric nucleic acid moleculesfurther comprising repeats of a regularly occurring monomer unit. Insome instances, the clonally-amplified single-stranded ordouble-stranded, multimeric nucleic acid molecules may be at least 0.1kb in length, at least 0.2 kb in length, at least 0.3 kb in length, atleast 0.4 kb in length, at least 0.5 kb in length, at least 1 kb inlength, at least 2 kb in length, at least 3 kb in length, at least 4 kbin length, at least 5 kb in length, at least 6 kb in length, at least 7kb in length, at least 8 kb in length, at least 9 kb in length, at least10 kb in length, at least 15 kb in length, or at least 20 kb in length,or any intermediate value spanned by the range described herein, e.g.,about 2.45 kb in length.

In some instances, the clonally-amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may comprisesingle-stranded or double-stranded multimeric nucleic acid moleculescomprising from about 2 to about 100 copies of a regularly repeatingmonomer unit. In some instances, the number of copies of the regularlyrepeating monomer unit may be at least 2, at least 3, at least 4, atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, and at least 100. In some instances, thenumber of copies of the regularly repeating monomer unit may be at most100, at most 95, at most 90, at most 85, at most 80, at most 75, at most70, at most 65, at most 60, at most 55, at most 50, at most 45, at most40, at most 35, at most 30, at most 25, at most 20, at most 15, at most10, at most 5, at most 4, at most 3, or at most 2. Any of the lower andupper values described in this paragraph may be combined to form a rangeincluded within the present disclosure, for example, in some instancesthe number of copies of the regularly repeating monomer unit may rangefrom about 4 to about 60. Those of skill in the art will recognize thatthe number of copies of the regularly repeating monomer unit may haveany value within this range, e.g., about 12. Thus, in some instances,the surface density of clonally-amplified target sequences in terms ofthe number of copies of a target sequence per unit area of the supportsurface may exceed the surface density of oligonucleotide primers evenif the hybridization and/or amplification efficiencies are less than100%.

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification reaction formulations mayyield increased clonal copy number compared to that obtained usingconventional supports and amplification protocols. In some instances,e.g., wherein the clonally-amplified target (or sample) oligonucleotidemolecules comprise concatenated, multimeric repeats of a monomerictarget sequence, the clonal copy number may be substantially smallerthan compared to that obtained using conventional supports andamplification protocols. Thus, in some instances, the clonal copy numbermay range from about 1 molecule to about 100,000 molecules (e.g., targetsequence molecules) per amplified colony. In some instances, the clonalcopy number may be at least 1, at least 5, at least 10, at least 50, atleast 100, at least 500, at least 1,000, at least 2,000, at least 3,000,at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least8,000, at least 9,000, at least 10,000, at least 15,000, at least20,000, at least 25,000, at least 30,000, at least 35,000, at least40,000, at least 45,000, at least 50,000, at least 55,000, at least60,000, at least 65,000, at least 70,000, at least 75,000, at least80,000, at least 85,000, at least 90,000, at least 95,000, or at least100,000 molecules per amplified colony. In some instances, the clonalcopy number may be at most 100,000, at most 95,000, at most 90,000, atmost 85,000, at most 80,000, at most 75,000, at most 70,000, at most65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000,at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most20,000, at most 15,000, at most 10,000, at most 9,000, at most 8,000, atmost 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000,at most 2,000, at most 1,000, at most 500, at most 100, at most 50, atmost 10, at most 5, or at most 1 molecule per amplified colony. Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the clonal copy number may range from about 2,000molecules to about 9,000 molecules. Those of skill in the art willrecognize that the clonal copy number may have any value within thisrange, e.g., about 2,220 molecules in some instances, or about 2molecules in others.

As noted above, in some instances the amplified target (or sample)oligonucleotide molecules (or nucleic acid molecules) may compriseconcatenated, multimeric repeats of a monomeric target sequence. In someinstances, the amplified target (or sample) oligonucleotide molecules(or nucleic acid molecules) may comprise a plurality of molecules eachof which comprises a single monomeric target sequence. Thus, the use ofthe disclosed low-binding supports alone or in combination withoptimized amplification reaction formulations may result in a surfacedensity of target sequence copies that ranges from about 100 targetsequence copies per mm² to about 1×10¹² target sequence copies per mm².In some instances, the surface density of target sequence copies may beat least 100, at least 500, at least 1,000, at least 5,000, at least10,000, at least 15,000, at least 20,000, at least 25,000, at least30,000, at least 35,000, at least 40,000, at least 45,000, at least50,000, at least 55,000, at least 60,000, at least 65,000, at least70,000, at least 75,000, at least 80,000, at least 85,000, at least90,000, at least 95,000, at least 100,000, at least 150,000, at least200,000, at least 250,000, at least 300,000, at least 350,000, at least400,000, at least 450,000, at least 500,000, at least 550,000, at least600,000, at least 650,000, at least 700,000, at least 750,000, at least800,000, at least 850,000, at least 900,000, at least 950,000, at least1,000,000, at least 5,000,000, at least 1×10⁷, at least 5×10⁷, at least1×10⁸, at least 5×10⁸, at least 1×10⁹, at least 5×10⁹, at least 1×10¹⁰,at least 5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least 1×10¹² ofclonally amplified target sequence molecules per mm². In some instances,the surface density of target sequence copies may be at most 1×10¹², atmost 5×10¹¹, at most 1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most5×10⁹, at most 1×10⁹, at most 5×10⁸, at most 1×10⁸, at most 5×10⁷, atmost 1×10⁷, at most 5,000,000, at most 1,000,000, at most 950,000, atmost 900,000, at most 850,000, at most 800,000, at most 750,000, at most700,000, at most 650,000, at most 600,000, at most 550,000, at most500,000, at most 450,000, at most 400,000, at most 350,000, at most300,000, at most 250,000, at most 200,000, at most 150,000, at most100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000,at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000,at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most10,000, at most 5,000, at most 1,000, at most 500, or at most 100 targetsequence copies per mm². Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the surface densityof target sequence copies may range from about 1,000 target sequencecopies per mm² to about 65,000 target sequence copies mm². Those ofskill in the art will recognize that the surface density of targetsequence copies may have any value within this range, e.g., about 49,600target sequence copies per mm².

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification buffer formulations mayresult in a surface density of clonally-amplified target (or sample)oligonucleotide molecules (or clusters) ranging from about from about100 molecules per mm² to about 1×10¹² colonies per mm². In someinstances, the surface density of clonally-amplified molecules may be atleast 100, at least 500, at least 1,000, at least 5,000, at least10,000, at least 15,000, at least 20,000, at least 25,000, at least30,000, at least 35,000, at least 40,000, at least 45,000, at least50,000, at least 55,000, at least 60,000, at least 65,000, at least70,000, at least 75,000, at least 80,000, at least 85,000, at least90,000, at least 95,000, at least 100,000, at least 150,000, at least200,000, at least 250,000, at least 300,000, at least 350,000, at least400,000, at least 450,000, at least 500,000, at least 550,000, at least600,000, at least 650,000, at least 700,000, at least 750,000, at least800,000, at least 850,000, at least 900,000, at least 950,000, at least1,000,000, at least 5,000,000, at least 1×10⁷, at least 5×10⁷, at least1×10⁸, at least 5×10⁸, at least 1×10⁹, at least 5×10⁹, at least 1×10¹⁰,at least 5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least 1×10¹²molecules per mm². In some instances, the surface density ofclonally-amplified molecules may be at most 1×10¹², at most 5×10¹¹, atmost 1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most1×10⁹, at most 5×10⁸, at most 1×10⁸, at most 5×10⁷, at most 1×10⁷, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm². Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedmolecules may range from about 5,000 molecules per mm² to about 50,000molecules per mm². Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 molecules per MM².

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification buffer formulations mayresult in a surface density of clonally-amplified target (or sample)oligonucleotide molecules (or clusters) ranging from about from about100 molecules per mm² to about 1×10¹² colonies per mm². In someinstances, the surface density of clonally-amplified molecules may be atleast 100, at least 500, at least 1,000, at least 5,000, at least10,000, at least 15,000, at least 20,000, at least 25,000, at least30,000, at least 35,000, at least 40,000, at least 45,000, at least50,000, at least 55,000, at least 60,000, at least 65,000, at least70,000, at least 75,000, at least 80,000, at least 85,000, at least90,000, at least 95,000, at least 100,000, at least 150,000, at least200,000, at least 250,000, at least 300,000, at least 350,000, at least400,000, at least 450,000, at least 500,000, at least 550,000, at least600,000, at least 650,000, at least 700,000, at least 750,000, at least800,000, at least 850,000, at least 900,000, at least 950,000, at least1,000,000, at least 5,000,000, at least 1×10⁷, at least 5×10⁷, at least1×10⁸, at least 5×10⁸, at least 1×10⁹, at least 5×10⁹, at least 1×10¹⁰,at least 5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least 1×10¹²molecules per mm². In some instances, the surface density ofclonally-amplified molecules may be at most 1×10¹², at most 5×10¹¹, atmost 1×10¹¹, at most 5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most1×10⁹, at most 5×10⁸, at most 1×10⁸, at most 5×10⁷, at most 1×10⁷, atmost 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, atmost 850,000, at most 800,000, at most 750,000, at most 700,000, at most650,000, at most 600,000, at most 550,000, at most 500,000, at most450,000, at most 400,000, at most 350,000, at most 300,000, at most250,000, at most 200,000, at most 150,000, at most 100,000, at most95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000,at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000,at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most5,000, at most 1,000, at most 500, or at most 100 molecules per mm². Anyof the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedmolecules may range from about 5,000 molecules per mm² to about 50,000molecules per mm². Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 molecules per mm².

In some instances, the use of the disclosed low-binding supports aloneor in combination with optimized amplification buffer formulations mayresult in a surface density of clonally-amplified target (or sample)oligonucleotide colonies (or clusters) ranging from about from about 100colonies per mm² to about 1×10¹² colonies per mm². In some instances,the surface density of clonally-amplified colonies may be at least 100,at least 500, at least 1,000, at least 5,000, at least 10,000, at least15,000, at least 20,000, at least 25,000, at least 30,000, at least35,000, at least 40,000, at least 45,000, at least 50,000, at least55,000, at least 60,000, at least 65,000, at least 70,000, at least75,000, at least 80,000, at least 85,000, at least 90,000, at least95,000, at least 100,000, at least 150,000, at least 200,000, at least250,000, at least 300,000, at least 350,000, at least 400,000, at least450,000, at least 500,000, at least 550,000, at least 600,000, at least650,000, at least 700,000, at least 750,000, at least 800,000, at least850,000, at least 900,000, at least 950,000, at least 1,000,000, atleast 5,000,000, at least 1×10⁷, at least 5×10⁷, at least 1×10⁸, atleast 5×10⁸, at least 1×10⁹, at least 5×10⁹, at least 1×10¹⁰, at least5×10¹⁰, at least 1×10¹¹, at least 5×10¹¹, or at least 1×10¹² coloniesper mm². In some instances, the surface density of clonally-amplifiedcolonies may be at most 1×10¹², at most 5×10¹¹, at most 1×10¹¹, at most5×10¹⁰, at most 1×10¹⁰, at most 5×10⁹, at most 1×10⁹, at most 5×10⁸, atmost 1×10⁸, at most 5×10⁷, at most 1×10⁷, at most 5,000,000, at most1,000,000, at most 950,000, at most 900,000, at most 850,000, at most800,000, at most 750,000, at most 700,000, at most 650,000, at most600,000, at most 550,000, at most 500,000, at most 450,000, at most400,000, at most 350,000, at most 300,000, at most 250,000, at most200,000, at most 150,000, at most 100,000, at most 95,000, at most90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000,at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000,at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most1,000, at most 500, or at most 100 colonies per mm².

Any of the lower and upper values described in this paragraph may becombined to form a range included within the present disclosure, forexample, in some instances the surface density of clonally-amplifiedcolonies may range from about 5,000 colonies per mm² to about 50,000colonies per mm². Those of skill in the art will recognize that thesurface density of clonally-amplified colonies may have any value withinthis range, e.g., about 48,800 colonies per mm².

In some cases the use of the disclosed low-binding supports alone or incombination with optimized amplification reaction formulations may yieldsignal from the amplified and labeled nucleic acid populations (e.g., afluorescence signal) that has a coefficient of variance of no greaterthan 50%, such as 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less than 5%.

In some cases, the support surfaces and methods as disclosed hereinallow amplification at elevated extension temperatures, such as at 15 C,20 C, 25 C, 30 C, 40 C, or greater, or for example at about 21 Cor 23 C.

In some cases, the use of the support surfaces and methods as disclosedherein enable simplified amplification reactions. For example, in somecases amplification reactions are performed using no more than 1, 2, 3,4, or 5 discrete reagents.

In some cases, the use of the support surfaces and methods as disclosedherein enable the use of simplified temperature profiles duringamplification, such that reactions are executed at temperatures rangingfrom a low temperature of 15 C, 20 C, 25 C, 30 C, or 40 C, to a hightemperature of 40 C, 45 C, 50 C, 60 C, 65 C, 70 C, 75 C, 80 C, orgreater than 80 C, for example, such as a range of 20 C to 65 C.

Amplification reactions are also improved such that lower amounts oftemplate (e.g., target or sample molecules) are sufficient to lead todiscernable signals on a surface, such as 1 pM, 2 pM, 5 pM, 10 pM, 15pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1,000 pM,2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM,9,000 pM, 10,000 pM or greater than 10,000 pM of a sample, such as 500nM. In exemplary embodiments, inputs of about 100 pM are sufficient togenerate signals for reliable signal determination.

Fluorescence imaging of support surfaces: The disclosed solid-phasenucleic acid amplification reaction formulations and low-bindingsupports may be used in any of a variety of nucleic acid analysisapplications, e.g., nucleic acid base discrimination, nucleic acid baseclassification, nucleic acid base calling, nucleic acid detectionapplications, nucleic acid sequencing applications, and nucleicacid-based (genetic and genomic) diagnostic applications. In many ofthese applications, fluorescence imaging techniques may be used tomonitor hybridization, amplification, and/or sequencing reactionsperformed on the low-binding supports.

Fluorescence imaging may be performed using any of a variety offluorophores, fluorescence imaging techniques, and fluorescence imaginginstruments known to those of skill in the art. Examples of suitablefluorescence dyes that may be used (e.g., by conjugation to nucleotides,oligonucleotides, or proteins) include, but are not limited to,fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof,including the cyanine derivatives Cyanine dye-3 (Cy3), Cyanine dye-5(Cy5), Cyanine dye-7 (Cy7), etc. Examples of fluorescence imagingtechniques that may be used include, but are not limited to,fluorescence microscopy imaging, fluorescence confocal imaging,two-photon fluorescence, and the like. Examples of fluorescence imaginginstruments that may be used include, but are not limited to,fluorescence microscopes equipped with an image sensor or camera,confocal fluorescence microscopes, two-photon fluorescence microscopes,or custom instruments that comprise a suitable selection of lightsources, lenses, mirrors, prisms, dichroic reflectors, apertures, andimage sensors or cameras, etc. A non-limiting example of a fluorescencemicroscope equipped for acquiring images of the disclosed low-bindingsupport surfaces and clonally-amplified colonies (or clusters) of targetnucleic acid sequences hybridized thereon is the Olympus IX83 invertedfluorescence microscope equipped with) 20×, 0.75 NA, a 532 nm lightsource, a bandpass and dichroic mirror filter set optimized for 532 nmlong-pass excitation and Cy3 fluorescence emission filter, a Semrock 532nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) where theexcitation light intensity is adjusted to avoid signal saturation.Often, the support surface may be immersed in a buffer (e.g., 25 mMACES, pH 7.4 buffer) while the image is acquired.

In some instances, the performance of nucleic acid hybridization and/oramplification reactions using the disclosed reaction formulations andlow-binding supports may be assessed using fluorescence imagingtechniques, where the contrast-to-noise ratio (CNR) of the imagesprovides a key metric in assessing amplification specificity andnon-specific binding on the support. CNR is commonly defined as:CNR=(Signal−Background)/Noise. The background term is commonly taken tobe the signal measured for the interstitial regions surrounding aparticular feature (diffraction limited spot, DLS) in a specified regionof interest (ROI). While signal-to-noise ratio (SNR) is often consideredto be a benchmark of overall signal quality, it can be shown thatimproved CNR can provide a significant advantage over SNR as a benchmarkfor signal quality in applications that require rapid image capture(e.g., sequencing applications for which cycle times must be minimized),as shown in the example below. As illustrated in FIGS. 6A and 6B, athigh CNR the imaging time required to reach accurate discrimination (andthus accurate base-calling in the case of sequencing applications) canbe drastically reduced even with moderate improvements in CNR. FIGS. 6Aand 6B provide simulation data for signal and background intensities(solid lines) and integrated average values (dashed lines) measured as afunction of CNR (CNR=1.25 in FIG. 6A and 12.49 in FIG. 6B) andintegration time (SNR=2 in both figures), which illustrate the improveddiscrimination that may be achieved by using CNR as the signal qualitymetric. FIG. 7 provides examples of the impact of improved CNR in imagedata on the imaging integration time required to accurately detectfeatures such as clonally-amplified nucleic acid colonies on the supportsurface.

In most ensemble-based sequencing approaches, the background term istypically measured as the signal associated with ‘interstitial’ regions(see FIG. 8). In addition to “interstitial” background (B_(inter))“intrastitial” background (B_(intra)) exists within the region occupiedby an amplified DNA colony. The combination of these two backgroundsignals dictates the achievable CNR, and subsequently directly impactsthe optical instrument requirements, architecture costs, reagent costs,run-times, cost/genome, and ultimately the accuracy and data quality forcyclic array-based sequencing applications. The B_(inter) backgroundsignal arises from a variety of sources; a few examples includeauto-fluorescence from consumable flow cells, non-specific adsorption ofdetection molecules that yield spurious fluorescence signals that mayobscure the signal from the ROI, the presence of non-specific DNAamplification products (e.g., those arising from primer dimers). Intypical next generation sequencing (NGS) applications, this backgroundsignal in the current field-of-view (FOV) is averaged over time andsubtracted. The signal arising from individual DNA colonies (i.e.,(S)−B_(inter) in the FOV) yields a discernable feature that can beclassified. In some instances, the intrastitial background (B_(intra))can contribute a confounding fluorescence signal that is not specific tothe target of interest, but is present in the same ROI thus making itfar more difficult to average and subtract.

As will be demonstrated in the examples below, the implementation ofnucleic acid amplification on the low-binding substrates of the presentdisclosure may decrease the B_(inter) background signal by reducingnon-specific binding, may lead to improvements in specific nucleic acidamplification, and may lead to a decrease in non-specific amplificationthat can impact the background signal arising from both the interstitialand intrastitial regions. In some instances, the disclosed low-bindingsupport surfaces, optionally used in combination with the disclosedhybridization and/or amplification reaction formulations, may lead toimprovements in CNR by a factor of 2, 5, 10, 100, or 1000-fold overthose achieved using conventional supports and hybridization,amplification, and/or sequencing protocols. Although described here inthe context of using fluorescence imaging as the read-out or detectionmode, the same principles apply to the use of the disclosed low-bindingsupports and nucleic acid hybridization and amplification formulationsfor other detection modes as well, including both optical andnon-optical detection modes.

The disclosed low-binding supports, optionally used in combination withthe disclosed hybridization and/or amplification protocols, yieldsolid-phase reactions that exhibit: (i) negligible non-specific bindingof protein and other reaction components (thus minimizing substratebackground), (ii) negligible non-specific nucleic acid amplificationproduct, and (iii) provide tunable nucleic acid amplification reactions.Although described herein primarily in the context of nucleic acidhybridization, amplification, and sequencing assays, it will beunderstood by those of skill in the art that the disclosed low-bindingsupports may be used in any of a variety of other bioassay formatsincluding, but not limited to, sandwich immunoassays, enzyme-linkedimmunosorbent assays (ELISAs), etc.

System: Provided herein are systems for performing base discriminationor base classification reactions using the surface described herein.Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to coupling the oligonucleotidemolecules, hybridizing sample or target nucleic acids to the attachedoligonucleotide molecules, and detecting or imaging the signal on thesurface.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to analyze the sequence of the nucleicacids in such sequencing techniques based on the detection offluorescent nucleotides or oligonucleotides. The detectioninstrumentation used to read the fluorescence signals on such arrays maybe based on either epifluorescence or total internal reflectionmicroscopy. One detection instrument has been proposed that use anoptical sequencing-by-synthesis reader. The reader can include a laserthat induces fluorescence from a sample within water channels of a flowcell. The fluorescence is emitted and collected by imaging optics whichcomprises one or more objective lens and tube lens. Optical imagersinclude, among other things, a light source to illuminate a sample inthe region of interest, one or more detectors, and optical components todirect light from the region of interest to the detector(s). The opticalimagers may also include a focus mechanism that maintains focus of theoptical components on the region of interest in order that lightreceived at the detectors is received in focus.

Method of Base Pair classification: Provided herein are methods ofperforming nucleic acid base pair discrimination or base pairclassification, the method comprising: a) providing a surface; whereinthe surface comprises: i) a substrate; ii) at least one hydrophilicpolymer coating layer; iii) a plurality of oligonucleotide moleculesattached to at least one hydrophilic polymer coating layer; and iv) atleast one discrete region of the surface that comprises a plurality ofclonally-amplified sample nucleic acid molecules annealed to theplurality of attached oligonucleotide molecules, wherein the pluralityof annealed clonally-amplified sample nucleic acid molecules are presentwith a surface density of at least 10,000 molecules/mm², b) performing anucleic acid amplification reaction on sample nucleic acid moleculesprior to or after annealing them to the plurality of oligonucleotidemolecules; and c) performing a cyclic series of single nucleotidebinding or incorporation reactions, wherein the nucleotides are labeledwith a detectable tag.

Provided herein also includes method of performing nucleic acidsequencing by utilizing the surfaces or system described herein. In someembodiments, the detectable tag is a fluorophore. In some embodiments,the detectable tag is Cyanine dye-3 (Cy3), and wherein a fluorescenceimage of the surface acquired using an Olympus IX83 invertedfluorescence microscope equipped with) 20×, 0.75 NA, a 532 nm lightsource, a bandpass and dichroic mirror filter set optimized for 532 nmlong-pass excitation and Cy3 fluorescence emission filter, a Semrock 532nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) undernon-signal saturating conditions while the surface is immersed in abuffer following the binding or incorporation of a first Cy3-labelednucleotide exhibits a contrast-to-noise (CNR) ratio of at least 20.

In some embodiments, the nucleic acid amplification reaction comprises abridge amplification reaction. In some embodiments, the nucleic acidamplification reaction comprises an isothermal bridge amplificationreaction. In some embodiments, the nucleic acid amplification reactioncomprises a rolling circle amplification (RCA) reaction. In someembodiments, the nucleic acid amplification reaction comprises ahelicase-dependent amplification reaction. In some embodiments, thenucleic acid amplification reaction comprises a recombinase-dependentamplification reaction. In some embodiments, the at least onehydrophilic polymer coating layer exhibits a water contact angle of lessthan 50 degrees.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1—Hydrophilic Substrates

Studies were performed to prepare and evaluate low non-specific bindingsupport surfaces using poly(ethylene glycol) (PEG) molecules ofdifferent molecular weight and functional end groups. One or more layersof PEG were linked to a glass surface via silane coupling to thefunctional end groups. Examples of functional groups that may be usedfor coupling include, but not limited to, biotin, methoxy ether,carboxylate, amine, NHS ester, maleimide, and bis-silane.Oligonucleotide primers with different base sequences and basemodifications were then tethered to the surface layer at variousdensities. Both surface functional group density and oligonucleotideconcentration were varied to target certain primer density ranges.Additionally, primer density can be controlled by dilutingoligonucleotide with other molecules that carry the same functionalgroup. For example, amine-labeled oligonucleotide can be diluted withamine-labeled polyethylene glycol in a reaction with NHS-ester coatedsurface to reduce the final primer density. Primers comprising differentlengths of a linker positioned between the hybridization region and thesurface attachment functional group can also be applied to controldensity. Example linkers include poly-T and poly-A strands (0 to 20bases in length) at the 5′ end of the primer, PEG linkers (3 to 20 unitsin length), and hydrocarbon-chain linkers of various lengths (e.g., C6,C12, C18, etc.). To measure the primer density, fluorescence-labeledprimers were immobilized to the surface and the fluorescence reading wascompared with that for a dye solution of known concentration.

Low non-specific binding (also referred to herein as “passivated”)substrate surfaces are desirable so that biomolecules, such as proteinand nucleic acids, do not “stick” to the surfaces. Examples of lownon-specific binding (low NSB) surfaces prepared using standardmonolayer surface preparations and various glass surface treatments areprovided below. Performing nucleic acid amplification successfully onpassivated surfaces creates a unique challenge. Since passivated,hydrophilic surfaces exhibit ultra-low NSB of proteins and nucleicacids, novel conditions must be utilized to achieve high passivation,improved primer deposition reaction efficiencies, hybridizationconditions, and induce effective nucleic acid amplification. Solid-phasenucleic acid hybridization and amplification processes require nucleicacid template attachment to the low binding or passivated surface, andsubsequent protein delivery and binding to the surface. A combination ofa new primer surface conjugation formulation (identified through Cy3oligonucleotide graft titration) and the resulting ultra-lownon-specific background (as evaluated using NSB functional test resultsfor red and green dyes) demonstrate the viability of these approaches.

In order to scale primer density and add additional dimensionality tohydrophilic or amphoteric surfaces, multi-layer coatings have beenapplied and tested using PEG and other hydrophilic polymers. By usinghydrophilic and amphoteric surface layering approaches that include, butare not limited to, the polymer/co-polymer materials described here, itis possible to increase primer loading on the support surfacesignificantly. Conventional PEG coated supports that use a monolayerprimer deposition process have been reported for single moleculesequencing applications, but they do not yield high copy numbers fornucleic acid amplification. Here, we disclose that “layering” can beaccomplished using traditional crosslinking approaches andchemistry-compatible monomer or polymer subunits such that one or morehighly-crosslinked layers can be built sequentially. Non-limitingexamples of polymers that are suitable for use include of streptavidin,poly acrilamide, polyester, dextram, poly-lysine, polyethylene glycol,and poly-lysine copolymers poly acrylamide, poly (N-isopropylacrylamide)(PNIPAM), poly(2-hydroxyethyl methacrylate), (PHEMA),poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA),polyester, dextran, poly-lysine, polyethylene glycol (PEG), polyacrylicacid (PAA), poly(vinylpyridine), poly(vinylimidazole) and poly-lysinecopolymers. The different layers may be attached to each other using anyof a variety of covalent or non-covalent reactions including, but notlimited to, biotin-streptavidin binding, azide-alkyne click reactions,amine-NHS ester reactions, thiol-maleimide reactions, and ionicinteractions between positively charged polymers and negatively chargedpolymers. It is also conceivable that these high primer densitymaterials can be constructed in solution and subsequently layered ontothe surface in multiple steps. With this approach, it is possible togenerate low NSB/low background substrate surfaces (FIGS. 9-13) forperforming solid-phase nucleic acid amplification and sequencingchemistries that provide significantly improved nucleic acidamplification, such that the signal-to-background ratios can be tuned tomeet the needs of a specific sequencing application (FIG. 14 and FIG.15). FIG. 9 provides an example of image data from a study to determinethe relative levels of non-specific binding of a green fluorescent dyeto glass substrate surfaces treated according to different surfacemodification protocols. FIG. 10 provides an example of image data from astudy to determine the relative levels of non-specific binding of a redfluorescent dye to glass substrate surfaces treated according todifferent surface modification protocols. FIG. 11 provides an example ofoligonucleotide primer grafting data for substrate surfaces treatedaccording to different surface modification protocols.

Method for Preparing a 2-Layer PEG Surface with Thiol-MaleimideChemistry:

A glass slide was cleaned using a 2M KOH treatment of 30 minutes at roomtemperature, washed, and then surface silanol groups were activatedusing an oxygen plasma. Silane-PEGSK-Thiol (Creative PEGWorks, Inc.,Durham, N.C.) was applied at a concentration of 0.1% in ethanol. After a2-hour coating reaction, the slide was washed thoroughly with ethanoland water, and then reacted with 2.5 mM of Maleimide-PEG-SuccinimidylValerate (MW=20K) in dimethylformamide (DMF) for 30 minutes. Theresulting surface was washed and promptly reacted with 5′-amine-labeledoligonucleotide primer at room temperature for 2 hours. Excesssuccinimidyl esters on the surface were deactivated by reacting with 100mM glycine at pH9 following the primer immobilization.

Method for Preparing a Multi-Layer PEG Surface with NHS Ester-AmineChemistry:

A glass slide is cleaned by 2M KOH treatment of 30 minutes at roomtemperature, washed, and then surface silanol groups are activated usingan oxygen plasma. Silane-PEG2K-amine (Nanocs, Inc., New York, N.Y.) isapplied at a concentration of 0.5% in ethanol solution. After a 2-hourcoating reaction, the slide was washed thoroughly with ethanol andwater. 100 uM of 8-arm PEG NHS (MW=10K, Creative PEGWorks, Inc., Durham,N.C.) was introduced at room temperature for 20 minute in a solventcomposition that can include 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90percent organic solvent and 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90percent low ionic strength buffer. The resulting surface was washed andreacted with 20 μM multi-arm PEG amine (MW=10K, Creative PEGWorks, Inc.,Durham, N.C.) for 2 hours. The resulting amine-PEG surface was thenreacted with a mixture of multi-arm PEG-NHS and amine-labeledoligonucleotide primer at varying concentrations. This process can berepeated to generate additional PEG layers on the surface.

Solid-Phase Isothermal Amplification:

In considering various isothermal amplification methods, it can be shownthat each isothermal amplification method has a unique optimum primerdensity range, which requires tunable surface coatings to maximize theamplification efficiency. In some cases higher primer surface densityscales with larger template copy numbers (FIG. 15). In some cases,although higher surface densities of primers may yield high foregroundsignals (i.e., sequence-specific signals) for some amplificationapproaches, they may lead to high background signals and thus provedetrimental for other amplification approaches. In FIG. 16, primerdeposition concentrations are shown at the top of the figure, while thevarious helicase isothermal amplification formulations tested areindicated in the images acquired of the resulting surface following theisothermal amplification reaction. The image for each surface wasexpected to appear red if specific amplification had occurred. As isevident from the series of images for formulation “58” in FIG. 16, asthe primer density was increased the color fades from red to green, thusindicating a reduction in specific amplification. In addition, it can beseen that including Betaine (a common buffer additive) in theamplification reaction formulation decreases the degree of nonspecificamplification in favor of specific amplification, thereby resulting inbrighter signals and improved CNR. The combination of low-binding,layered support surfaces, tunable surface densities of primers,improvements in hybridization and/or amplification reaction formulations(including adjustments in buffer components and additives (e.g., choiceof buffer, pH, solvent, ionic strength, detergents, formamide, betaine,crowding agents and other additives, etc.), and resulting improvementsin amplification rates and specificity should lead to unprecedentedimprovements in next-generation sequencing of nucleic acids.

The present disclosure addresses the challenges of achieving highlymonoclonal amplification of library nucleic acid strands on hydrophilicsubstrates for a variety of applications that require signalenhancement, such as nucleic acid detection, sequencing, and diagnosticsapplications. Conventional isothermal methods for nucleic acidamplification for generating monoclonal clusters of a library nucleicacid strand are limited and have flaws. Examples of their performancelimitations include long clustering times (e.g., 2+ hours), therequirement for high temperature (e.g., 60 C or above), the inability toamplify/cluster efficiently on certain surfaces, high cost,polyclonality issues, reagent stability issues, etc. Despite the wealthof isothermal amplification methods described in the literature, thereare only one or two methods which have been successfully applied tocommercial sequencing applications. Here, we propose isothermalamplification strategies that successfully generate clusters ofmonoclonal copies of a library DNA fragment (or other nucleic acid) forapplications such as DNA sequencing and eliminate or alleviate theaforementioned problems.

The design criteria for developing ensemble DNA amplification/DNAsequencing composition changes to decrease background signals (B_(inter)and B_(intra)) and facilitate controlled DNA amplification on lowbinding substrates included: (i) decreased nonspecific DNA amplification(e.g., due to amplification of primer dimers) in interstitial regions(B_(inter)) as compared to traditional/prior art approaches, (ii)decreased amounts of non-specific DNA amplification product (e.g.,primer dimers) within specific DNA colonies (B_(intra)) as compared totraditional/prior art approaches, (iii) increased control of specificDNA amplification (e.g., reaction times, cycle times, primer surfacedensity titration, primer surface density, primer sequence, etc.) on lowbinding substrates, such that signal-to-background (S/B) ratio isreduced even in the absence of hybridization and amplificationformulation improvements.

Example 2—Helicase-Dependent Amplification on Low Binding Surfaces withImproved Specificity

It is well known that helicase-dependent amplification is highly proneto non-specific amplification, such as primer dimer formation. We mayreduce this non-specific amplification on the surface through anycombination of following methods: (i) designing oligonucleotide primersthat produces less primer dimer, (ii) adjust primer surface density onthe multilayered support surface, (iii) performing the reaction athigher temperatures using thermophilic enzymes, (iv) using amplificationbuffer additives such as those mentioned above and non-self-primingprimer sequences in combination, and (v) introducing one or more fullnucleic acid denaturation and primer hybridization steps.

Specific Helicase-Dependent Amplification on Low NSB Surfaces in theAbsence of SSB Protein:

Helicase-dependent amplification of linear template strands can beachieved with reduced non-specific amplification and highly-efficientclonal amplification on low binding surfaces. A forward primer on thesurface gets extended on a single-stranded template library strand usinga polymerase and helicase-containing amplification reaction mixture.Then, optionally, the template strand is denatured and washed away.Alternatively, the duplex may be unwound by the helicase activity.Either method leaves the forward strand extended from the surfaceconjugated primer, partially or fully in single-stranded form.Subsequently, a surface-tethered reverse primer hybridizes to thisforward strand and is also extended, thereby creating a double-strandedbridge structure. The helicase present in the reaction mixture unwindsthe intermediate double-stranded amplicon strands which are thenavailable to re-hybridize to other free surface-tethered primers forsubsequent amplification rounds. For this to happen, the degree ofunwinding doesn't need to be extensive—just sufficient to convert primerhybridization regions into single stranded components (end fraying) toallow hybridization of subsequent surface-conjugated primers. Thehelicase used in this reaction should be capable of initiating unwindingfrom the ends of the bridge structure, and may be either a 3′ to 5′helicase or a 5′ to 3′ helicase. In some cases, it may be a superfamily1, 2, 3 4, 5 or 6 helicase. In some cases, it may be a highly processivehelicase (i.e., able to unwind many consecutive base pairs withoutreleasing the single-stranded or double-stranded structure) or ahelicase with limited processivity. Certain mutants of superfamily 1helicases that exhibit higher processivity, such as the UvrD303 mutantof helicase UvrD, may be used in this amplification scheme.

In order to facilitate unwinding by 5′ to 3′ helicases, all or a portionof one or both surface-tethered primers may include modifications tocreate specific loading sites for the 5′ to 3′ helicase. On a templatenucleic acid extended from such a primer, the modification site wouldact as a polymerase stop point thereby leaving the stretch of primersequence between the surface conjugation point and the modification sitealways in single-stranded form. This stretch would act as loading sitefor helicases with 5′ to 3′ directionality, as many helicases have muchbetter single stranded nucleic acid binding affinity, directing andenhancing the 5′ to 3′ helicase unwinding activity where it is neededfor helicase-dependent amplification on the support surface. Examples ofprimer modifications that may be used include, but are not limited to,an insertion of a PEG chain into the backbone of the primer between twonucleotides towards the 5′ end, insertion of an abasic nucleotide (i.e.,a nucleotide that has neither a purine nor a pyrimidine base), or alesion site which can be bypassed by the helicase.

Many helicases have co-factor proteins and specific conformations thatactivate or enhance the helicase activity. Examples include, but are notlimited to, the RepD protein for the PcrA helicase from Bst, the phi Xgene protein A for the E. coli Rep helicase, and the MutL protein forthe UvrD helicase. Addition of these accessory proteins may enable thedesired unwinding activity more effectively and thus further facilitatehelicase-dependent isothermal amplification. Some of these cofactorshave specific binding sequences or moieties which may be added to theprimers to direct the unwinding activity.

Helicase amplification formulations showed diminished non-specificamplification when using a mesophilic helicase and a strand displacingpolymerase formulation that lacked single stranded binding (SSB) protein(e.g., internal reference formulation #58). Unlike the case for mosthelicase-dependent amplification approaches, the exclusion of singlestranded binding protein (which typically is used to disrupt transientnon-specific hybridization) from the formulation was observed to reducenon-specific amplification. A variety of formulation changes were shownto mitigate the increase in non-specific hybridization on low bindingsurfaces.

Formulation composition change for improved helicase-dependentamplification: Additives such as Betaine are commonly known to decreasethe non-specific amplification for isothermal amplification reactionsperformed in solution. The criteria become more restrictive as highprimer surface densities are required to support tunable amplificationand high copy number in template nucleic acid colonies. At high primerdensity, non-specific amplification begins to abet the benefits of hightemplate copy number in the resulting colonies (FIG. 16). As a result,additional additive formulations have been discovered which abet thenon-specific amplification within a template nucleic acid colony. In theexample shown in FIG. 16, it can be clearly seen that the addition ofBetaine to formulation #58 creates more specific amplification(indicated by the red color) on higher primer density surfaces. Inaddition to betaine, it is possible to combine many different reactionformulation components to achieve higher amplification specificity insolution and on a low binding surface (FIG. 17 and FIG. 18).

Organic solvents such as acetonitrile, DMSO, DMF, ethanol, methanol andsimilar compounds alter the structure of single stranded and doublestranded nucleic acids through a process of oligonucleotide dehydration.Compounds such as 2-pyrrolidone and formamide are known to reduce themelting temperature of nucleic acids, and reduce secondary structurefrom high GC content oligonucleotides. Crowding agents are also known tostabilize nucleic acid structure. In low pH buffers, hybridization andhydrogen bonding in base pairing become more favorable. When combiningthe different attributes of each of these respective formulations, it ispossible to increase specific annealing of oligonucleotide sequences bymore than 2 orders-of-magnitude over traditional approaches. Bycombining these compounds and adding them to the amplificationformulations described below, non-specific amplification, such asarising from primer dimers, is drastically abated, and good yields ofspecific amplification product are still observed in solution (FIG. 17)and on the disclosed low NSB surfaces (FIG. 18).

Example 3—Modified Rolling Circle Multiple Displacement Amplification(Modified RCA-MDA)

Nucleic acid library fragments are ligated to adapter sequences (thatcontain forward, reverse, and sequencing primers, and anyidentification/barcode sequences) and circularized either in thesolution, or on the low binding surface.

Circularized ssDNA is then captured by or hybridized to a forwardsurface primer either in solution or on the surface and extended in aRCA reaction by a strand displacing polymerase which makessingle-stranded concatemeric copies of the library nucleic acid andadapter sequences. Reverse primers hybridize to this concatemericforward template at multiple positions and are extended by the RCAreaction mix, thereby making concatemeric reverse copies. During thisprocess reverse strands are displaced by each other. An upstream reversestrand extension would displace the downstream extension, thus creatingsingle-stranded concatemeric reverse strands. The addition of helicasescan generate single-stranded regions of nucleic acid that last longenough to restart the hybridization and trigger displacement cascades,which can increase the amplification copy number in a relativelycontrolled fashion. Alternatively, the addition of recombinases andaccessory proteins can hybridize primers into the homologous regions ofduplexed DNA in a process called strand invasion. This will restartdisplacement and hybridization cascades, and increases the copy numberin the colony.

Copy number in the RCA-MDA colonies is determined by the primer surfacedensity, which dictates how frequently and successfully the initialconcatemers or displaced concatemers are hybridized with the forward andthe reverse primers. Increased primer density on low binding surfaceshas proven to generate higher amplification copy numbers in theseclusters (FIG. 15). In summary, it is possible to increase the copynumber or specific amplification, and decrease the non-specificamplification on low binding surfaces, using one or a combination of thefollowing methods: (i) specific copy number may be increased byincreasing the efficiency of primer template hybridizations throughformulation changes (FIG. 16), (ii) specific copy number may beincreased by increasing the primer density on low binding substrates(FIG. 14 and FIG. 15), (iii) non-specific amplification of primer dimersor chimeric DNA generation may be decreased by using the additivesdescribed above, (iv) amplification incubation temperatures may beincreased using thermostable enzymes combined with formulation changesas previously described to reduce the non-specific amplification, (v)primer compositions that comprise non-self-hybridizing primer sequencesmay be used in combination with additives and/or increased amplificationincubation temperatures to decrease non-specific primer dimeramplification.

Example 4—Single-Stranded DNA Binding (SSB) Protein Mediated IsothermalAmplification

SSB proteins can resolve secondary structures in nucleic acid strands,stabilize the single-stranded DNA after unwinding, prevent or disrupttransient non-extensive hybridization of two nucleic acid strands (aprecursor to primer dimer amplification), facilitate specifichybridization of short oligonucleotides to the correct complementaryregions in a target oligonucleotide, and interact and direct enzymes tothe forked junctions of single-stranded and double-stranded nucleicacids. C-terminal truncation mutations in SSB were reported to removethe cooperative binding to ssDNA while its monomers exhibit strongerbinding to ssDNA and reduce the melting temperature of dsDNA. Forexample, a C-terminal truncation mutation of a phage SSB protein, T4gp32, yields a protein (gp32ΔC) that reduces the melting temperature ofdsDNA by tens of degrees compared to the performance of the wild typeprotein.

In this amplification scheme, we use these proteins in a formulationthat comprises both truncated and wild type SSB proteins (and stranddisplacing polymerase) to transiently melt the ends of thedouble-stranded nucleic acid bridge intermediates and allow surfaceprimer hybridization and primer extension. Even though SSB slows downnucleic acid hybridization, it is known to facilitate specifichybridization in general. Hence, the use of truncated SSB proteins suchas T4 gp32ΔC, for example, could enable more efficient hybridization ofthe 3′ ends in the bridged nucleic acid structures to other freesurface-tethered primers. While this would also disrupt the freshlyformed primer template complexes, an optimized formulation should allowextension of such complexes by a strand displacing polymerase. SSBproteins are found in a variety of phages (for example, T4 gp32),bacteria, archaea, fungi, and eukaryotic organisms. Some arethermostable SSB proteins, of which the C-terminal truncated forms couldenable more efficient nucleic acid end melting at optimized highertemperatures, thus enable SSB-dependent thermophilic amplification whiletaking advantage of lower non-specific amplification at highertemperature.

Through the use of additives such as those mentioned above, primersequence design and specific hybridization and/or amplificationformulations, and optimized temperatures for use with appropriatetemperature- and chemically-resistant enzymes and proteins, thisamplification method can be tailored to drive highly-specificamplification, while eliminating non-specific amplification.

This scheme can be used to amplify circular DNA as well as linear DNA,where each initiation of extension by a strand displacing polymerase canlead to multiple subsequent multiple displacement amplification eventsthat yield concatemeric copies of the circular DNA template (see thesection on modified RCA-MDA above).

We have found that SSB-mediated amplification (using, for example, phi29SSB) of circular library DNA is much faster than traditional RCA (e.g.,requiring amplification times of only 30 min to 1 hour vs 2 to 3 hoursfor traditional RCA). Products of SSB-mediated amplification performedin solution ran as discrete concatemeric ladders on gels.

Example 5—Low-Temperature Thermal Cycling Bridge Amplification on LowBinding Surfaces

By using combinations of additives for improved hybridization and/oramplification formulations, thermostable SSB proteins, and/or truncatedSSB proteins, PCR formulations have been developed that can be thermallycycled at lower temperatures than the traditional methods outlined inMullis' original PCR disclosure. In this scheme, it is possible to useadditives to drive nucleic acid hybridization and de-hybridizationtemperatures below their traditional values. For example, formamide iscommonly used to reduce the melt temperature (T_(m)) of DNA andsubsequent DNA de-hybridization can be performed at a temperature ofaround 60 degrees. On the other hand, re-annealing temperaturestypically also require temperature ramps from high temperatures (95degrees C.) to close to room temperature. It is possible to createformulations such that the re-annealing temperature stringencies can bedrastically reduced. Using such a formulation would constitute asignificant improvement over traditional bridge amplification methods,such that temperature ramps can be performed between 20 and 60 degrees.Decreases in temperature ramp requirements and improved hybridizationstringency on low binding substrates could yield the followingadvantages over traditional bridge amplification: (i) decreasedamplification times; (ii) simplified instrumentation design, (iii)decreased reagent usage through faster and more specific hybridizationresulting in more efficient amplification rates, and (iv) reducedreagent costs. It is also possible to show that the stringency of theimproved hybridization would decrease the number of amplification cyclesrequired for amplification on the support surface.

FIGS. 19A-B provide examples of data that illustrate the improvements inhybridization efficiency that may be obtained using the low non-specificbinding supports and improved hybridization formulations of the presentdisclosure (FIG. 19A) as compared to those for a conventionalhybridization formulation (FIG. 19B).

FIG. 20 illustrates a workflow for nucleic acid sequencing using thedisclosed low binding supports and hybridization/amplification reactionformulations of the present disclosure, and non-limiting examples of theprocessing times that may be achieved thereby.

Example 6—Preparation of 2-Layer PEG Surface with Thiol-MaleimideChemistry

A glass slide is chemically treated to remove organics and activatehydroxyl groups for silane coupling (various methods include plasmatreatment, piranha etching, base wash, base baths, high temperatureglass annealing and any combination thereof. Silane-PEG5K-Thiol(Creative PEGWorks, Inc) is applied at concentration of 0.1% in Ethanolsolution. After 2-hour of coating reaction, the slide is washedthoroughly with Ethanol and water and then reacted with 2.5 mM ofMaleimide-PEG-Succinimidyl Valerate (MW 20K) in DMF for 30 minutes. Theresulted surface is washed and promptly reacted with 5′-amine-labeledoligonucleotide primer at room temperature for 2 hours. The excesssuccinimidyl esters on surface are deactivated with 100 mM of glycine atPH9 after the primer immobilization. This approach confers negligiblelow binding solid support surfaces through and efficient primer andpolymer iterative coupling that exceeds traditional methodologies for byalmost 2 orders of magnitude.

Example 7 Preparation of Multi-Layer PEG Surface with NHS Ester-AmineChemistry

A glass slide is chemically treated to remove organics and activatehydroxyl groups for silane coupling (various methods include plasmatreatment, piranha etching, base wash, base baths, high temperatureglass annealing and any combination thereof. Silane-PEG-amine (Nanocs,Inc) is applied at concentration of 0.1%-2% in clean Ethanol solution.After 2-hour of coating reaction, the slide is washed thoroughly withEthanol and water. Multi-arm PEG NHS is introduced at room temperaturefor 5-30 minute in a solvent composition that can include 5, 10, 20, 30,40, 50, 60, 70, 80 or 90 percent organic solvent and 5, 10, 20, 30, 40,50, 60, 70, 80 or 90 percent low ionic strength buffer. The resultedsurface is washed and reacted with multi-arm PEG amine (MW 10k, CreativePEGWorks, Inc). The resulted amine-PEG surface is then reacted with amixture of multi-arm PEG NHS and amine-labeled oligonucleotide primer atvarying concentrations. This process can be repeated to generateadditional PEG layers on surface. This approach confers negligible lowbinding solid support surfaces through and efficient primer and polymeriterative coupling that exceeds traditional methodologies for by almost2 orders of magnitude.

Example 8—Calculation of CNR on Cluster Data

Typically, when fluorescence detection is used for solid-phase assays,signals are generated by tethering and/or amplifying molecules, coupledwith or followed by attachment of reporter dye molecules. This processproduces both specific and non-specific signals. The non-specificcomponents are commonly referred to as non-specific noise or background(arising from either inter- or intra-stitial contributions) whichinterferes with the measurement of the specific signal and reducescontrast-to-noise ratio (CNR).

Non-specific background can be generated either from dye moleculesadsorbed non-specifically to the support surface, or from non-specificamplification of, for example, primer-dimer pairing on the surface. Bothmechanisms produce substantial fluorescence background when fluorescentreporters are used to label the specific molecules of interest.

The disclosed low-binding support surfaces and associated methods foruse demonstrate significant improvement in minimizing the non-specificbackground. As shown in the examples described below, estimatednon-specific background is well below 10% of the total signal using thespecified amplification method and support surface. On the other hand,conventional amplification methods and support surfaces often producebackground signals that are 30% to 50% of the total signal.

Note that, as used herein, the non-specific background or noise is onlyone component of the total system noise, which may also include othercontributions from the detection system, such as photon shot noise,auto-fluorescence background, image sensor noise, illumination noise(e.g., arising from fluctuations in illumination intensity), etc. Inthis regard, it may be possible to extend the disclosed approaches forCNR improvement through novel support surfaces and associatedhybridization and amplification methods to achieve even largerimprovements for NGS and other bioassay technologies by, for example,correcting for signal impurities that may arise from traditionalsequencing-by-synthesis, such as through the use of pre phasing andphasing, and also correcting for errors incurred through the loss of DNAstrands and/or DNA damage arising from stringent wash conditions orde-blocking (reversible terminator removal) over multiple assay reactioncycles.

In general, the assay for measuring CNR for a solid-phase bioassayincludes the steps of:

(1) preparing the disclosed low binding substrates to be functionalizedwith receptor, target, and/or capture oligonucleotides of interest.

(2) capturing the receptor, target, and/or capture oligonucleotides,which may be directly labeled or may be a precursor to a subsequentlabeling reaction. If no amplification or additional probe-labeling stepis required, one proceeds to step 5. If a probe labeling step isrequired to create a reporter, one proceeds to step 4. Otherwise, oneproceeds to step 3.

(3) perform amplification of the receptor, target, and/or captureoligonucleotides via traditional immunoassay signal amplification,oligonucleotide replication amplification (e.g., using bridge,isothermal, RCA, HDA, or RCA-MDA amplification strategies).

(4) probe the amplified target with a reporter label (e.g., through theuse of a fluorescent species or other type of reporter). This step isapplicable to any surface-based bioassay including, but is not limitedto, genotyping, nucleic acid sequencing, or surface-basedtarget/receptor identification.

(5) perform an appropriate detection methodology. For fluorescenceimaging, the detection methodology can be configured in various ways.For example, traditional optical microscopy methods would include all ora subset of the following components: illumination or excitation lightsource, objective, sample, other optical components (such as a tubelens, optical filters, dichroic reflectors, etc.), and a detectionmodality (e.g., using an EMCCD camera, CCD camera, sCMOS, CMOS, PMT, APDor other traditional method for measuring light levels). Fornon-light-based detection, electrical signals can be measured usingvarious means including, but not limited to, field effect transistor(FET) detection, electrode-based measurement of electrical signals(direct or alternating), tunneling currents, measurement of magneticsignals, etc.

The use of imaging and signal processing from a specified field-of-view(FOV) to calculate the CNR is illustrated in FIG. 8, whereCNR=(Signal−Background)/(Noise), and whereBackground=(B_(intrastitial)+B_(interstitial)) as illustrated in thefigure.

For the following examples of the calculation of CNR forclonally-amplified clusters of nucleic acid sequences on the low-bindingsupports of the present disclosure, an image analysis program was usedto find representative foreground bright spots (“clusters”). Typically,spots are defined as a small connected region of image pixels thatexhibit a light intensity above a certain intensity threshold. Onlyconnected regions that comprise a total pixel count that falls within aspecified range are counted as spots or clusters. Regions that are toobig or too small in terms of the number of pixels are disregarded.

Once a number of spots or clusters have been identified, the averagespot or foreground intensity and other signal statistics are calculated,for example, the maximum, average, and/or interpolated maximumintensities may be calculated. The median or average value of all spotintensities is used to represent spot foreground intensity.

A representative estimate of the background region intensity may bedetermined using one of several different methods. One method is todivide images into multiple small “tiles” which each include, e.g.,25×25 pixels. Within each tiled region, a certain percentage of thebrightest pixels (e.g., 25%) are discarded, and intensity statistics arecalculated for the remaining pixels. Another method for determiningbackground intensity is to select a region of at least 500 pixels, orlarger, which is free of any foreground “spots” (as defined in theprevious step), and then to calculate intensity statistics. For eitherof these methods, a representative background intensity (median oraverage value) and standard deviation are then calculated. The standarddeviation of the intensity in the selected regions is used as therepresentative background variation.

Contrast-to-noise ratio (CNR) is then calculated as (foregroundintensity−background intensity)/(background standard deviation).

FIGS. 21-23 provide examples of raw image data and intensity datahistograms used to calculate CNR for difference combinations of nucleicacid amplification methodology and the low-binding supports describedhere. In each of these examples, the upper histogram is the backgroundpixel intensity histogram, the lower histogram is the foreground spotintensity histogram, and a portion of the original image is alsoincluded. Note that the images are not on the same intensity scale, sovisual brightness perception does not indicate actual intensity.

For these examples, low binding solid supports were created using themethods previously discussed. Oligonucleotide primers (one or two primersequences depending on the amplification scheme used) were grafted usingthe disclosed methods at varying densities. Surface densities for eachof these experiments were estimated to be approximately 100Kprimers/μm². Primer surface density was estimated using the followingmethodology: (i) a fluorescence titration curve was prepared using a GETyphoon (GE Healthcare Lifesciences, Pittsburgh, Pa.) and a capillaryflow cell of known area (40 mm²), height (0.5 mm), and volume (200 μl)containing known concentrations of Cy3-dCTP, (ii) the primers grafted tothe low-binding support were hybridized to Cy3-labeled complementaryoligonucleotides using a conventional hybridization protocol (3× salinesodium citrate (SSC) at 37 degrees C. or at room temperature (RT);hybridization conditions should be characterized for completeness),fluorescence intensity for the resulting signal on the surface wasmeasured using the same GE Typhoon instrument used to generate thecalibration curve, (iii) and the number of primer molecules tethered perunit area of surface was calculated based on a comparison of themeasured surface signal to the calibration curve.

DNA library sequences were then hybridized to the tethered primers. Thehybridization protocols used for the library hybridization step can varydepending on surface properties, but controlled library input isrequired to create resolvable DNA amplified colonies.

DNA amplification was performed for this example using the followingprotocols: (i) bridge amplification@ 28 cycles with primer density ofapproximately 1K primers/um², (ii) bridge amplification@ 28 cycles withhigher primer density>5K primers/um², and (iii) rolling circleamplification (RCA) for 90 minutes with primer density of approximately2-4 K primers/um².

Post amplification, the amplified DNA was hybridized with acomplementary “sequencing” primer and a sequencing reaction mixcomprising a Cy3-labeled dNTP was added (“first base” assay) todetermine the first base CNR for each of the respective methodologies.The sequencing reaction mixture used for the “first base assay” caninclude any combination of labeled nucleotides, such that 4 bases can bediscriminated, an enzyme that incorporates the modified nucleotidetriphosphate (dNTP), and a relevant incorporation buffer, metal cationsand cofactors, etc.

Following first base incorporation, the sequencing reaction mixture wasexchanged with buffer, imaging was performed using the same GE Typhooninstrument, and CNR was calculated on the resulting images.

FIG. 21 provides an example of fluorescence image and intensity data fora low-binding support of the present disclosure on which solid-phasenucleic acid amplification was performed using bridge amplification @ 28cycles with primer density of approximately 2K primers/um² to createclonally-amplified clusters of a template oligonucleotide sequence. Inthis example, the background intensity was 592 counts (with a standarddeviation of 66.5 counts), the foreground intensity was 1047.3 counts,and the calculated CNR=(1047.3−592)/66.5=455.3/66.5=6.8. The estimatednon−specific noise=(592−100)/(1047−100)=52%.

FIG. 22 provides a second example of fluorescence image and intensitydata for a low-binding support of the present disclosure on whichsolid-phase nucleic acid amplification was performed using bridgeamplification @ 28 cycles with higher primer density>5K primers/um² tocreate clonally-amplified clusters of a template oligonucleotidesequence. In this example, the background intensity was 680 counts (witha standard deviation of 118.2 counts), the foreground intensity was 1773counts, and the calculated CNR=(1773−680)/118.2=1093/118.2=9.2. Theestimated non−specific noise=(680−100)/(1773−100)=35%.

FIG. 23 provides an example of fluorescence image and intensity data fora low-binding support of the present disclosure on which solid-phasenucleic acid amplification was performed using rolling circleamplification (RCA) for 90 minutes with primer density of approximately100 K primers/um² to create clonally-amplified clusters of a templateoligonucleotide sequence. In this example, the background intensity was254 counts (with a standard deviation of 22.7 counts), the foregroundintensity was 6161 counts, and the calculatedCNR=(6161−254)/22.7=5907/22.7=260. Note the dramatic improvement in CNRachieved through the use of this combination of low-binding surface andamplification protocol. The estimated non-specificnoise=(254−100)/(6161−100)=3%.

Example 9—Modification of Polymer Support Surfaces

Modification of a surface for the purposes disclosed herein involvesmaking surfaces reactive against many chemical groups (—R), includingamines. When prepared on an appropriate substrate, these reactivesurfaces can be stored long term at room temperature for example for atleast 3 months or more. Such surfaces can be further grafted with R-PEGand R-primer oligomer for on-surface amplification of nucleic acids, asdescribed elsewhere herein. Plastic surfaces, such as cyclic olefinpolymer (COP), may be modified using any of a large number of methodsknown in the art. For example, they can be treated with Ti: Sapphirelaser ablation, UV-mediated ethylene glycol methacrylate photografting,plasma treatment, or mechanical agitation (e.g., sand blasting, orpolishing, etc.) to create hydrophilic surfaces that can stay reactivefor months against many chemical groups, such as amines. These groupsmay then allow conjugation of passivation polymers such as PEG, orbiomolecules such as DNA or proteins, without loss of biochemicalactivity. For example, attachment of DNA primer oligomers allows DNAamplification on a passivated plastic surface while minimizing thenon-specific adsorption of proteins, fluorophore molecules, or otherhydrophobic molecules.

Additionally, surface modification can be combined with, e.g., laserprinting or UV masking, to create patterned surfaces. This allowspatterned attachment of DNA oligomers, proteins, or other moieties,providing for surface-based enzymatic activity, binding, detection, orprocessing. For example, DNA oligomers may be used to amplify DNA onlywithin patterned features, or to capture amplified long DNA concatemersin a patterned fashion. In some embodiments, enzyme islands may begenerated in the patterned areas that are capable of reacting withsolution-based substrates. Because plastic surfaces are especiallyamenable to these processing modes, in some embodiments as contemplatedherein, plastic surfaces may be recognized as being particularlyadvantageous.

Furthermore, plastic can be injection molded, embossed, or 3D printed toform any shape, including microfluidic devices, much more easily thanglass substrates, and thus can be used to create surfaces for thebinding and analysis of biological samples in multiple configurations,e.g., sample-to-result microfluidic chips for biomarker detection or DNAsequencing.

Specific localized DNA amplification on modified plastic surfaces havebeen achieved that produced spots with an ultra-high contrast to noiseratio and very low background when probed with fluorescent labels.

We have grafted a representative hydrophilized and amine reactive cyclicolefin polymer surface with amine-primer and amine-PEG and found that itsupports rolling circle amplification. We then discovered that whenprobed with fluorophore labeled primers, or when labeled dNTPs added tothe hybridized primers by a polymerase, bright spots of DNA ampliconswere observed that exhibited signal to noise ratios greater than 100with backgrounds that are extremely low, indicating highly specificamplification, and ultra-low levels of protein and hydrophobicfluorophore binding which are hallmarks of the high accuracy detectionsystems such as fluorescence-based DNA sequencers.

Here a plastic flow cell is tested that is populated with tethered DNAclusters and probed for the 1st base of the library sequence. Forpopulation of the surface with DNA, a hydrophilized cyclic olefinpolymer (COP) plastic surface was grafted with 25-mer amine-primer 1,amine-primer 2, and amine-5K PEG as for PEG-NHS coated glass surfaces,as previously described in Examples 1 and 7 and elsewhere herein. 5 pMof a circularized DNA library that contains primer 2 sequence,sequencing primer sequence and a sequence that is complementary toprimer 1, in addition to the library insert, was then hybridized to thesurface for 15 minutes. Rolling Circle Amplification (RCA) was thenperformed as described in Examples 2-5 and elsewhere herein, for thecreation of concatemeric sequence DNA coils of up to 0.5-1 Mb in length.The sequencing primer was hybridized, and thelst base was incorporatedusing a fluorophore labeled dNTP set with a polymerase to label clusterswith 3 different colors as shown in FIG. 25A.

A parallel experiment using identical parameters, starting with a glass,rather than a COP surface (with surface preparation as described inExample 7) was carried out in order to provide a comparison betweenpassivated glass and passivated COP surfaces. As shown in FIGS. 25A-B,the signal produced by incorporation of the first fluorescently labeledbase on COP surfaces is comparable to that obtained on similarly treatedglass surfaces, both in terms of the intensity and the resolution of theobserved spots. This suggests that the methods disclosed herein providea general method for the preparation of surfaces for the immobilization,amplification, and detection of nucleic acids.

Intensity and CNR are determined for both glass and plastic. One sees atFIG. 26A that both glass and plastic exhibit an intensity of signalunder detection conditions that is substantially above background. Forboth glass and plastic, intensity of signal is at left and background isdepicted at right. One sees at FIG. 26B that CNR for both glass andplastic are above 50 under the conditions assayed.

Example 10—Surface Production

A surface exhibiting negligible nonspecific binding to organic dyes andproteins, exhibiting stability up to at least 95 C, chemical stabilityto High pH (0.1 M NaOH), Low pH (>5.0); organic solvents (Methanol,Ethanol, Acetonitrile, Formamide, oxidants, phosphines), long termstorage stability, low input library requirements and scalable primerloading is produced as follows. The process comprises cleaning andsilanizing or passivating the surface.

The surface is washed using a 2M KOH solution in combination with analconox/hellmanex detergent and rinsed using ethanol. The surface isthen heated to 560 C to expose OH groups. Surfaces are alternately or incombination subjected to a plasma treatment.

Surfaces are silanized using 5 mg/mL Silane-SkPEG-NHS (99.9%Ethanol/0.01% Acetic acid) and heated to 65 C, and tested using aKOH/Detergent/Heat cleaned surface. Alternately or in combination,surfaces are silanized using 10 mg/mL Silane-SkPEG-NHS (90% DMF/10% 100mM MES PH5.5) and heated to 25 C, and tested using KOH/Detergent/Heatcleaned surface or with a plasma treated surface.

A number of dyes are compatible with these surfaces, such as Cy3-C,R11-U, Cy3.5C, 647N-A, Cy5-G, 660-U, Cy5.5-C (Note: Only dye at 200 nM).An exemplary dye mix comprises Cy3-A, Cy3.5-C, Cy5-U, AHO690-G.

Such surfaces are tuneably loaded with primers, at low concentrations(5.0×10⁴ primers/um²), at high concentrations (1.0×10⁷ primers/um²), andat concentrations of values within a range defined by these endpoints oroutside of this range.

Concentration is optionally measured as follows. Make Cy3-dCTP solutionof different concentrations, measure the FL intensity with a GE Typhoon(GE Healthcare Lifesciences, Pittsburgh, Pa.) or suitable instrument ina capillary with fixed dimension (0.5 mm×5 mm or other area). Thisyields the primer loading when area is known and number of molecules isknown.

Concentrations, in primers/um² of 80,000; 160,000; 320,000; 640,000;1,300,000; 2,600,000; and 5,100,000 have been measured using thisapproach, and other concentrations of values within a range defined bythese endpoints or outside of this range are readily attainable. Thesedensities are facilitated by the presence of multilayer PEG or othersurfaces as disclosed herein.

Surfaces were seen to show no significant reduction in stability overone week of storage.

Densities were measured for a number of surface variants and resultswere as seem below. 3-layer multi-arm PEG (8,16,8) on PEGamine-APTES,exposed to two layers of 7 uM primer pre-loading, exhibited aconcentration of 2,000,000 to 10,000,000 on the surface. Similarconcentrations were observed for 3-layer multi-arm PEG (8,16,8) and(8,64,8) on PEGamine-APTES exposed to 8 uM primer, and 3-layer multi-armPEG (8,8,8) using star-shape PEG-amine to replace dumbbell-shaped 16merand 64mer.

Using these approaches, it was observed that increased primer densitiesyielded higher foreground intensities, higher colony densities andhigher CNR. For example, a 10 pM input yielded a CNR of 10 on a surfacehaving a Primer Density<1.0×10⁴ primer/um² while a similar input yieldeda CNR of 40-60 at a Primer Density>1.0×10⁶ primer/um².

Example 11—High CNR Surfaces Yield High Quality Data

Current state of the art surfaces, and low and high CNR surfaces such asthose disclosed herein were tested as to their fluorescence as detectedat a first channel and a second channel, corresponding to a first and asecond dye.

One observed that, with increasing CNR, one sees a clearer resolution ofindividual detection events. These detection events align along distinctaxes corresponding to dye emission spectra, rather than to higher error‘clouds’ as seen in the top three files of FIG. 27. Turning to thebottom three files of FIG. 27, this more accurate data collectionmanifests itself as narrower, higher peaks at specific expectedwavelengths and fewer data points at intermediate positions. This moreclearly resolved dataset translates into more accuratefluorescence-based base calls resulting from assays performed on highCNR surfaces.

Example 12—Clonally-Amplified, Multimeric Target OligonucleotideMolecules

FIG. 28 provides a schematic illustration of a clonally-amplified,multimeric target oligonucleotide sequence hybridized to a surfacecomprising a high surface density of oligonucleotide adapter or primermolecules such as, for example, 4,000 or more molecules per um2 (left)and to a surface comprising a lower surface density such as, forexample, below 500 molecules per um2 of oligonucleotide adapter orprimer molecules (right) and illustrates the resulting improvement inCNR that may be achieved. Several surfaces were prepared to haveoligonucleotide density that is higher than the 2000 molecules/uM², andthe fluorescence image of the surfaces has a contrast to noise ratio ofgreater than 20.

Example 13—Reduction in Input Nucleic Acid Requirements

FIG. 29 provides a comparison of the experimental outcomes forperforming a traditional hybridization reaction on a low-binding supportsurface of the present disclosure and performing an optimizedhybridization reaction on the low-binding support surface of the presentdisclosure. Traditional hybridization approaches use SSC buffer andheating to 95 degrees and then a slow 2 hour cool. After attachingoligonucleotide primers to the low-binding support surface, efficienthybridization of target nucleic acids to the oligonucleotide primers maysuffer from diminished collisional frequencies on the low bindingsurface. Due, at least in part, to decreased coupling of captureoligonucleotides on prior art surfaces, traditional hybridizationmethods for adding target DNA to surface-bound primers requires inputDNA concentrations of up to 10 nM (see FIG. 29, left, showing binding oflabeled target oligonucleotide to binding support surfaces). Even atthese high concentrations, coupling of target oligonucleotides islimited. In comparison, a non-complementary oligonucleotide at the samerespective concentrations was used as a negative control (bottom rowFIG. 29) By comparison, using new hybridization reaction conditions thathave been developed, which include a mixture comprising PEG, a solventwith a decreased polarity as compared to water, such as ethanol,methanol, isopropanol, acetonitrile, butanol, or the like, formamide anda low pH buffer (<7) target nucleic acid sequences were hybridized tosurfaced-attached oligonucleotides at input concentrations of the targetnucleic acid as low as 50 pM. The drop in the target nucleic acid inputconcentration indicates a roughly 200-fold increase in hybridizationefficiency (see FIG. 29, right, showing the binding of labeled targetoligonucleotide to the disclosed low-binding surfaces), which providesthe disclosed low-binding support surfaces with a significant advantagefor use in sequencing technologies where input library DNA may bescarce. The efficient primer coupling process and hybridizationconditions can allow preparation of a surface having a low nonspecificbinding and high surface density of oligonucleotide primers that wouldnot be achieved using traditional primer coupling chemistries orhybridization conditions described in the art. In comparison, anon-complementary oligonucleotide at the same respective concentrationswas used as a negative control (bottom row FIG. 29, right) Thedetectable tag for each of the images is Cyanine dye-3 (Cy3), andwherein a fluorescence image of the surface acquired) 20×, 0.75 NA, a532 nm light source, a bandpass and dichroic mirror filter set optimizedfor 532 nm long-pass excitation and Cy3 fluorescence emission filter, aSemrock 532 nm dichroic reflector, and a camera (e.g., an Andor sCMOS,Zyla 4.2)

Traditional or standard conditions were tested with hybridizationreporter probe (complementary oligonucleotide sequences labeled with aCy™3 fluorophore at the 5′ end) in 2×-5× saline-sodium citrate (SSC)buffer (std) at concentrations reported at 90 degree with a slow coolprocess (2 hours) to reach 37 degrees. The surfaces used for bothtesting conditions were ultra-low non-specific binding surfaces having alevel of non-specific Cy3 dye absorption of less than about 0.25molecules/μm2. Wells were washed with 50 mM Tris pH 8.0; 50 mM NaCl.Images were obtained acquired using an inverted microscope (OlympusIX83) equipped with 100×TIRF objective, NA=1.4 (Olympus), dichroicmirror optimized for 532 nm light (Semrock, Di03-R532-t1-25x36), abandpass filter optimized for Cy3 emission, (Semrock, FF01-562/40-25),and a camera (sCMOS, Andor Zyla) under non-signal saturating conditionsfor 1 s, (Laser Quantum, Gem 532, <1 W/cm2 at the sample) while sampleis immersed a buffer (25 mM ACES, pH 7.4 buffer). Conditions 50% ACN+MESwith 1 um oligonucleotide graft concentration and 25% ACN+MES+20%PEG+10% formaldehyde with 5.1 uM oligonucleotide graft concentrationwere chosen to test the viability of these conditions to improveexisting standard surface hybridization protocols on a low bindingsubstrate. The oligonucleotide probe was added at concentrationsspecified and hybridization performed for 2 min at 50 degrees C. Imageswere collected as described above and results shown in the figure.

Example 14—Comparison of Traditional Oligo Primer Coupling Chemistries,Hybridization Reaction Conditions, and Amplification Techniques onLow-Binding Support Surfaces

FIGS. 30-32 provide comparisons of the experimental outcomes forperforming traditional hybridization reactions or improved hybridizationreactions using the methodologies outlined in description of FIG. 29 onthe low-binding supports of the present disclosure where the oligoprimers were attached using a conventional coupling chemistry, which forNHS—NH2 coupling reactions are typically performed in sodium bicarbonatebuffer at pH=8.3. or an improved coupling chemistry, which entailchanging the polarity of the coupling buffer (organic based solvent)with the addition of a buffering component of pH>8.0, and where thehybridization reactions were followed by performing either RCA or Bridgeamplification. Amplification of target DNA on low-binding supportshaving an oligonucleotide primer surface density of less than a distinctsurface density threshold, either by PCR-based (“bridge”) or by rollingcircle amplification, gives elongated or spread target molecules thatcreates two challenges: 1) diminished packing densities, and 2)diminished signal. As a result, scaling a system for high throughputsequencing applications based on such a surface is drastically impaired.FIG. 30 shows the results of both PCR amplification (“bridge”; right)and rolling circle amplification (left) on low binding surfaces usingtraditional oligonucleotide coupling chemistries, such that theoligonucleotide density<1000 oligonucleotides/um2. In these images,labeled amplified target DNA can be seen assuming extended conformationsthat would present severe difficulties for imaging in sequencingapplications. By comparison, FIG. 31 shows the results of PCR/“bridge”amplification and FIG. 32 shows the results of rolling circleamplification (RCA) on surfaces having oligonucleotide primer surfacedensities of at least 1,000 molecules per um². These surfaces supportthe compaction of the amplified target DNA into highly localized regionsthat yield high fluorescence intensities from attached labels, and smallpixel areas in imaging. The high fluorescence intensities, notably, leadto increased signal when calculating spot intensities for sequencingapplications. Importantly, the enhanced contrast-to-noise ratioresulting from the use of the low-binding surfaces disclosed herein is afunction of both the very high signal resulting from this compaction ofthe amplified target DNA, and the very low background provided by thehydrophilic coated surfaces. Each of these images has a labelednucleotide with individual labels, such that each nucleotide has adifferent emission label. As an example, the labels may consist of Cy3,Cy3.5, Cy5, and Cy5.5 respectively and then imaged on an Olympus IX83microscope equipped with a 20×, 0.75 NA, a 532 nm light source, abandpass and dichroic mirror filter set optimized for 532 nm long-passexcitation and Cy3 fluorescence emission filter, a Semrock 532 nmdichroic reflector, and a camera (Andor sCMOS, Zyla 4.2) at an exposuretime of at least 0.5 s

Example 15—Comparison of Amplification Reactions on Low-Binding SupportSurfaces

FIG. 33 provides non-limiting examples of fluorescence images of atraditional support surface and a low-binding support surface of thepresent disclosure to which target oligonucleotides have been hybridizedand amplified. In order to facilitate sequencing accuracy, eachamplified target nucleic acid must be clearly separable from othertarget nucleic acids in images of the support surface. Each targetnucleic acid must also, during the sequencing cycle, present a signal(such as a fluorescence signal) that is related to identification ofeach nucleotide in the sequence (the process referred to as“base-calling”). This base-calling signal, which in many cases is simplythe fluorescence intensity provided by a label attached to the targetnucleic acid molecules, must be clearly and accurately resolvable aboveboth noise (variation in signal within a spot or target) and background(spurious signal generated nonspecifically due to characteristics of thematerial forming the experimental milieu). The ratio between contrastand noise (“CNR”) defines the ability to accurately determine which baseis present at each position in a target nucleic acid sequence, as wellas the read length, reproducibility, and throughput of a sequencingsystem. The presently disclosed low-binding support surfaces provide forreduced nonspecific protein and dye binding, thereby resulting in lowerbackground signal, and a more compact, brighter foreground signal,thereby yielding enhanced CNR and facilitating superior base-callingresults in sequencing applications. FIG. 32 shows a comparison between aPEG-coated surface prepared generally according to conventional methodsand adapted as necessary for the binding of clonally-amplified targetDNA (top, “Traditional); and the low-binding support surfaces of thepresent disclosure (top, “Element Current”). It is clear from the imagesthat the presently disclosed surfaces show notably sharper, more intensespots for measurement of clonally-amplified DNA than the conventionalsurface. Quantitative measurement of both spot intensity (fluorescenceattributable to clonally-amplified target DNA, equivalent to thesequencing signal) and background intensity show that the CNR for thedisclosed low-binding support surfaces (bottom, “Element Current Best”)far exceeds that achievable using the conventional surface (bottom,Traditional”), even under ideal imaging conditions such as when usingimproved hybridization conditions for the binding of target DNA (bottom,‘Traditional Improvement”). This increase in CNR is greater than whatcould reasonably be expected using a conventional support surface andrepresents both a qualitative and quantitative improvement over priorart surfaces. These improvements are achieved by generating a supportsurface that meets several criteria, e.g., reduced nonspecific proteinand dye binding, attachment of the correct density of oligonucleotidesto the surface, and hybridization/binding of a clonally-amplified targetnucleic acid to the surface to yield images with very high CNR thatenable enhanced base-calling in sequencing applications.

Example 16—Prophetic Example of Preparing Low-Binding Supports UsingOther Polymers

A glass slide is physically- or chemically-treated (e.g., using a plasmatreatment, a piranha cleaning step, an acid wash, a base wash, hightemperature glass annealing, or any combination thereof) to removeorganic contaminants and activate surface hydroxyl groups for silanecoupling. The prepared glass surface is then reacted with a silane tocovalently attached a first layer of functional groups (e.g., primaryamines) and/or a hydrophilic polymer layer. In some instances, forexample, a silane such as (3-aminopropyl)trimethoxysilane (APTMS) or(3-aminopropyl)triethoxysilane (APTES) 3 (3-acrylopropyl)trimethoxysilane is reacted with the surface using standard protocols tocovalently attach primary amine functional groups to the surface. Inother instances, a silane-modified polymer, e.g., a hydrophilic,heterobifunctional polymer comprising a silyl group at one end and asecond functional group (e.g., a primary amine, carboxyl group, etc.) atthe other end may be reacted directly with the surface (e.g., bycontacting the clean glass surface with the silane-modified polymer atconcentration of 0.1%-2% in ethanol for about 1 to 2 hours followed byrinsing with ethanol and water). Examples of suitable silane-modifiedpolymers include, but are not limited to, silane-PEG-NH2 (having apolyethylene glycol (PEG) molecular weight of, for example, 1000, 2000,3400, 5000, or 10K Daltons), silane-PEG-COOH (having a PEG molecularweight of, for example, 1000, 2000, 3400, 5000, or 10K Daltons),silane-PEG-maleimide (having a PEG molecular weight of, for example,1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-biotin (having a PEGmolecular weight of, for example, 1000, 2000, 3400, 5000, or 10KDaltons), silane-PEG-acrylate (having a PEG molecular weight of, forexample, 1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-silane(having a PEG molecular weight of, for example, 1000, 2000, 3400, 5000,or 10K Daltons), silane-modified polypropylene glycols (PPGs) of variousmolecular weights that comprise an additional functional group,silane-modified poly(vinyl alcohols) (PVAs) of various molecular weightsthat comprise an additional reactive functional group, silane-modifiedpolyethylenimine (PEIs) of various molecular weights that comprise anadditional reactive functional group, silane-modified poly(lysine) ofvarious molecular weights that comprise an additional reactivefunctional group, and the like, or any combination thereof.

In some instances, at least one additional layer of a hydrophilicpolymer layer is coupled to, or deposited on, the glass surfacefollowing the initial reaction of the surface with a silane orsilane-modified polymer. Any of a number of hydrophilic polymers knownto those of skill in the art including, but not limited to, polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine),poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide,poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA),poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA),poly-lysine, poly-glucoside, streptavidin, dextran, or any combinationthereof may be used where, in the case of covalent coupling, polymer(s)comprising the appropriate monofunctional, homobifunctional, and/orheterobifunctional reactive groups are selected for compatibility withthe chosen conjugation chemistry. In some cases, a derivatized polymeris used, such as a PEG-amine, a PEG-NHS, or a PEG-Acrylate. In somecases, a bifunctional PEG derivative is used, such as anacrylate-PEG-NHS. In some cases, these additional hydrophilic polymerlayers may be coupled to, or deposited on, the previous layer bycontacting the surface with a 0.1%-2% polymer in ethanol or anethanol/aqueous buffer solution for about 5 minutes to about 1 hour atroom temperature, followed by rinsing with ethanol or an ethanol/aqueousbuffer.

In some instances, a second, third, fourth, fifth, or more additionallayers of a hydrophilic polymer may be coupled to, or deposited on, theinitial layer of the support surface. In some instances, the polymermolecules within a layer may be cross-linked with each other usingappropriate homofunctional or heterofunctional cross-linking reagents.In some instances, the polymer molecules in different layers may becross-linked with each other. In some instances, one or more of thehydrophilic polymer layers may comprise a branched polymer, e.g., abranched PEG, branched poly(vinyl alcohol) (branched PVA), branchedpoly(vinyl pyridine), branched poly(vinyl pyrrolidone) (branched PVP),branched), poly(acrylic acid) (branched PAA), branched polyacrylamide,branched poly(N-isopropylacrylamide) (branched PNIPAM), branchedpoly(methyl methacrylate) (branched PMA), branched poly(2-hydroxylethylmethacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol)methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid(branched PGA), branched poly-lysine, branched poly-glucoside, brancheddextran, or any combination thereof.

One of more of the hydrophilic polymer layers may comprise a pluralityof covalently-attached oligonucleotide adapter or primer molecules,wherein the oligonucleotide molecules are covalently coupled to thepolymer using any of a variety of suitable conjugation chemistries knownto those of skill in the art. In some instances, the oligonucleotideadapter or primer molecules are covalently coupled to the polymer insolution, i.e., prior to coupling or depositing the polymer on thesurface. In some instances, the oligonucleotide adapter or primermolecules are covalently coupled to the polymer after it has beencoupled to or deposited on the surface. In some instances, at least onehydrophilic polymer layer comprises a plurality of covalently-attachedoligonucleotide adapter or primer molecules. In some instances, at leasttwo, at least three, at least four, or at least five layers ofhydrophilic polymer comprise a plurality of covalently-attached adapteror primer molecules.

The choice of polymer(s) used, the number of layers, the degree ofcross-linking within and between layers, the number of layers comprisingcovalently-attached oligonucleotide adapter or primer molecules, and thelocal concentration or surface density of oligonucleotide adapter orprimer molecules may be individually or collectively adjusted to “tune”the properties of the surface to achieve a desired surface wettability(as indicated, for example, by a water contact angle of less than 50degrees), a desired surface stability under prolonged exposure tosequencing/genotyping reagents and repeated thermocycling, which oftenrequire temperature ramps at a peak temperature of at least 95 degreesC. and held for at least 5 minutes and cycled multiple times of at least30 cycles, and a desired surface density of oligonucleotide adapter orprimer molecules (e.g., at least 1,000 adapter or primer molecules perμm²), which in turn provide for extremely low non-specific binding ofdye molecules or other labeled sequencing/genotyping reagents, improvedhybridization efficiency, improved amplification efficiency andspecificity, optimal densities of clonally-amplified target sequences(in terms of the number of clonal colonies per unit area, the number ofcopies of target sequence per unit area, or the number of amplifiedtarget molecules per unit area), higher contrast-to-noise ratios (CNRs)in images (e.g., fluorescence images) of the support surface (e.g.,CNR>20), and ultimately, improved detection accuracy or base-callingaccuracy in genotyping and sequencing applications.

Example 17—Acrylate Coupled Surfaces

Plasma treated, KOH treated, or plasma/KOH treated glass surfaces orsilicon wafers, or plasma treated COP surfaces were treated with(3-acrylopropyl) trimethoxysilane followed by incubation withbifunctional acrylate-PEG-NHS with average PEG molecular weights varyingfrom 1K to 6K, including especially PEG-3.4K. Molecular weights of500-10K were contemplated, with the limitation that the PEG had to besoluble in water at 42° C., soluble in water at 37° C., soluble in waterat room temperature, in a liquid state at 42° C., in a liquid state at37° C., or in a liquid state at room temperature. The PEG incorporationwas optionally assisted by the addition of up to 0.5% (w/w)2-hydroxy-2-methyl propiophenone, followed by UV treatment (10 minutesat 3.0 mW/cm2). Acrylate-PEG-NHS was used at concentrations of 3 mM and6 mM, with superior results being achieved with 6 mM acrylate-PEG-NHS.After washing to remove unbound polymer, surfaces were incubated at roomtemperature for a sufficient time to allow the autolysis of the NHSgroups, leaving free terminal carboxylate groups on the bound PEGmolecules. These surfaces were then activated by treatment withEDAC-HCl, and 5′-amino oligonucleotides were added to yield theoligonucleotide conjugated surface. A combination of SP6 oligonucleotide(25NT) and SPSP oligonucleotide (a 25NT with a 3′ phosphate cap) wereadded in a 1:1 ratio, or a 1:2, 1:5, or 1:10 ratio (SP6 oligonucleotideis a primer for surface-grown rolling circle amplification, while SPSPhelps to prevent random priming events or nonspecificcondensation/binding of RCA amplified products). After washing with 90%EtOH in MES at pH 9 to remove unbound oligonucleotide, a storage bufferwas added comprising ACES/KCFEDTA/Tween20. Surfaces may be stored for atleast 7 days under this buffer condition.

Surfaces comprising attached primers were then used for on-surfacerolling circle amplification of target nucleic acids, yielding condensednucleic acid molecules as shown elsewhere herein. Pre-prepared RCAproducts were also bound to the surface, yielding similarly compactnucleic acid structures.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in any combination in practicing the invention.It is intended that the following claims define the scope of theinvention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. (canceled)
 2. The method of claim 30, wherein the detectable tag is afluorophore.
 3. A method of performing nucleic acid sequencedetermination, the method comprising: (a) providing a surface, whereinthe surface comprises: (i) a substrate; (ii) at least one hydrophilicpolymer coating layer on the substrate; and (iii) a plurality ofoligonucleotide molecules attached to at least one hydrophilic polymercoating layer, wherein the plurality of oligonucleotide molecules ispresent with a surface density of at least 1,000 molecules per squaredmicrometer (μm²); and (b) contacting a plurality of sample nucleic acidmolecules with the plurality of oligonucleotide molecules attached tothe at least one hydrophilic polymer coating layer; and (c) performingat least a nucleotide binding reaction or a nucleotide incorporationreaction with a nucleotide that is labeled with a detectable tag and asample nucleic acid molecule of the plurality of sample nucleic acidmolecules or derivative thereof, wherein an image of the surfaceexhibits a contrast-to-noise ratio of at least
 20. 4. The method ofclaim 30, further comprising amplifying the plurality of sample nucleicacid molecules.
 5. The method of claim 4, wherein amplifying comprises abridge amplification reaction.
 6. The method of claim 4, whereinamplifying comprises a rolling circle amplification (RCA) reaction. 7.The method of claim 4, wherein amplifying comprises a helicase-dependentamplification reaction or a recombinase-dependent amplificationreaction.
 8. The method of claim 30, wherein the at least onehydrophilic polymer coating layer exhibits a water contact angle of lessthan 50 degrees.
 9. The method of claim 30, wherein the at least onehydrophilic polymer coating layer, comprises a molecule selected fromthe group consisting of polyethylene glycol (PEG), poly(vinyl alcohol)(PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylicacid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM),poly(methyl methacrylate) (PMA), poly(-hydroxylethyl methacrylate)(PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate)(POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside,streptavidin, and dextran.
 10. The method of claim 30, wherein the atleast one hydrophilic polymer coating layer comprises PEG.
 11. Themethod of claim 30, comprising a second hydrophilic polymer coatinglayer.
 12. The method of claim 30, wherein at least one hydrophilicpolymer coating layer comprises a polymer having a molecular weight ofat least 1,000 Daltons.
 13. The method of claim 30, wherein at least onehydrophilic polymer layer comprises a branched hydrophilic polymerhaving at least 4 branches.
 14. A method of performing nucleic acidsequence determination, the method comprising: (a) providing a surface,wherein the surface comprises: (i) a substrate; (ii) at least onehydrophilic polymer coating layer on the substrate; and (iii) aplurality of oligonucleotide molecules comprising a sequence thatincludes a polymerase stop point, such plurality attached to the atleast one hydrophilic polymer coating layer, and present with a surfacedensity of at least 1,000 molecules/μm²; (b) contacting a plurality ofsample nucleic acid molecules with the plurality of oligonucleotidemolecules attached to the at least one hydrophilic polymer coatinglayer; and (c) performing at least a nucleotide binding reaction or anucleotide incorporation reaction with a nucleotide that is labeled witha detectable tag and a sample nucleic acid molecule of the plurality ofsample nucleic acid molecules.
 15. The method of claim 30, whereincontacting in (b) is performed by contacting the surface with theplurality of sample nucleic acid molecules at a concentration of nogreater than 100 nanomolar (nM).
 16. The method of claim 30, whereincontacting in (b) is performed by contacting the surface with theplurality of sample nucleic acid molecules at a concentration of nogreater than 10 nM.
 17. The method of claim 30, wherein the plurality ofsample nucleic acid molecules are clonally-amplified prior to contactingto the plurality of oligonucleotide molecules.
 18. The method of claim30, wherein at least one sample nucleic acid of the plurality of samplenucleic acid molecules comprises a single-stranded multimeric nucleicacid molecule comprising repeats of a regularly occurring monomer unit.19. The method of claim 18, wherein the at least one sample nucleic acidof the plurality of sample nucleic acid molecules comprises adouble-stranded monomeric copy of the regularly occurring monomer unit.20. The method of claim 30, wherein a surface density of the pluralityof sample nucleic acid molecules is greater than the surface density ofthe plurality of oligonucleotide molecules.
 21. The method of claim 30,wherein said surface is positioned on an interior of a flow channel,flow cell, or capillary lumen.
 22. The method of claim 21, wherein theflow channel, flow cell, or capillary lumen are configured for use inperforming a nucleic acid hybridization, amplification, or sequencingreaction, or any combination thereof.
 23. The method of claim 30,wherein the surface comprising (i)-(iii) is at least one discrete regionof the surface, and wherein a background fluorescence intensity measuredat a region of the surface that is laterally-displaced from the at leastone discrete region is no more than twice of an intensity measured atthe at least one discrete region.
 24. A method of performing nucleicacid sequence determination, the method comprising: (a) providing asurface, wherein the surface comprises: (i) a substrate; (ii) at leastone hydrophilic polymer coating layer on the substrate, wherein the atleast one hydrophilic polymer coating layer comprises: (1) a first layercomprising a first monolayer of polymer molecules tethered to a surfaceof the substrate; (2) a second layer comprising a second monolayer ofpolymer molecules tethered to the polymer molecules of the firstmonolayer; and (3) a third layer comprising a third monolayer of polymermolecules tethered to the polymer molecules of the second monolayer,wherein the polymer molecules of at least one of the first monolayer,the second monolayer, and the third monolayer comprises branched polymermolecules; and (iii) a plurality of oligonucleotide molecules attachedto the at least one hydrophilic polymer coating layer, wherein theplurality is present with a surface density of at least 1,000 moleculesper squared micrometer (μm²); (b) contacting a plurality of samplenucleic acid molecules with the plurality of oligonucleotide moleculesattached to the at least one hydrophilic polymer coating layer; and (c)performing at least a nucleotide binding reaction or a nucleotideincorporation reaction with a nucleotide that is labeled with adetectable tag and a sample nucleic acid molecule of the plurality ofsample nucleic acid molecules or derivative thereof.
 25. The method ofclaim 24, wherein the second layer or the third layer further comprisesoligonucleotides tethered to the polymer molecules of the secondmonolayer or third monolayer, and wherein the oligonucleotides tetheredto the polymer molecules of the second monolayer or the third monolayerare distributed at a plurality of depths throughout the second layer orthe third layer.
 26. The method of claim 30, wherein the substratecomprises glass or plastic.
 27. The method of claim 24, wherein theplurality of the sample nucleic acid molecules is present at a surfacedensity of at least 10,000 molecules per squared millimeter (mm²). 28.The method of claim 30, further comprising extending an oligonucleotideof the plurality of oligonucleotide molecules by one nucleotide, whereinthe oligonucleotide is coupled to the sample nucleic acid molecule. 29.The method of claim 30, wherein in (c), the nucleotide is incorporated.30. A method of performing nucleic acid sequence determination, themethod comprising: (a) providing a surface, wherein the surfacecomprises: (i) a substrate; (ii) at least one hydrophilic polymercoating layer on the substrate; and (iii) a plurality of oligonucleotidemolecules attached to the at least one hydrophilic polymer coatinglayer, wherein the plurality is present with a surface density of atleast 1,000 molecules/μm²; (b) contacting a plurality of sample nucleicacid molecules with the plurality of oligonucleotide molecules attachedto the at least one hydrophilic polymer coating layer; and (c)performing at least a nucleotide binding reaction or a nucleotideincorporation reaction with a nucleotide that is labeled with adetectable tag and a sample nucleic acid molecule of the plurality ofsample nucleic acid molecules or derivative thereof, wherein afluorescence image of the surface exhibits a contrast-to-noise (CNR)ratio of at least 20 when the detectable tag is Cyanine dye-3 (Cy3) andwhen the fluorescence image of the surface is acquired using an invertedfluorescence microscope and a camera under non-signal saturatingconditions while the surface is immersed in a buffer.
 31. The method ofclaim 24, wherein the at least one hydrophilic polymer coating layerexhibits a water contact angle of less than 50 degrees.